Zika virus rna vaccines

ABSTRACT

The disclosure relates to tropical diseases such as viral mosquito borne illnesses and the treatment thereof. The invention includes ribonucleic acid vaccines and combination vaccines, as well as methods of using the vaccines and compositions comprising the vaccines for treating and preventing tropical disease.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/674,585, filed Aug. 11, 2017, which is a continuation ofinternational application number PCT/US2016/058324, filed Oct. 21, 2016,which claims the benefit under 35 U.S.C. § 119(e) of U.S. provisionalapplication No. 62/244,937, filed Oct. 22, 2015, U.S. provisionalapplication No. 62/247,347, filed Oct. 28, 2015, U.S. provisionalapplication No. 62/244,814, filed Oct. 22, 2015, U.S. provisionalapplication No. 62/247,390, filed Oct. 28, 2015, U.S. provisionalapplication No. 62/245,207, filed Oct. 22, 2015, U.S. provisionalapplication No. 62/247,445, filed Oct. 28, 2015, U.S. provisionalapplication No. 62/244,950, filed Oct. 22, 2015, U.S. provisionalapplication No. 62/247,595, filed Oct. 28, 2015, U.S. provisionalapplication No. 62/351,255, filed Jun. 16, 2016 and U.S. provisionalapplication No. 62/245,031, filed Oct. 22, 2015, each of which isincorporated by reference herein in its entirety.

BACKGROUND

Insects such as mosquitoes cause significant human suffering bytransmission of infectious disease to humans. The infections carried bymosquitoes afflict humans, as well as companion animals such as dogs andhorses. Infectious agents transmitted by mosquitos cause illnesses suchas encephalitis, Chikungunya, yellow fever, West Nile fever, malaria,and Dengue. The transmission of diseases associated with mosquito bitescan be interrupted by killing the mosquitoes, isolating infected peoplefrom all mosquitoes while they are infectious or vaccinating the exposedpopulation.

Deoxyribonucleic acid (DNA) vaccination is one technique used tostimulate humoral and cellular immune responses to foreign antigens,such as Malaria, JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and YFVantigens. The direct injection of genetically engineered DNA (e.g.,naked plasmid DNA) into a living host results in a small number of itscells directly producing an antigen, resulting in a protectiveimmunological response. With this technique, however, comes potentialproblems, including the possibility of insertional mutagenesis, whichcould lead to the activation of oncogenes or the inhibition of tumorsuppressor genes.

SUMMARY

Provided herein are ribonucleic acid (RNA) vaccines that build on theknowledge that RNA (e.g., messenger RNA (mRNA)) can safely direct thebody's cellular machinery to produce nearly any protein of interest,from native proteins to antibodies and other entirely novel proteinconstructs that can have therapeutic activity inside and outside ofcells. The RNA (e.g., mRNA) vaccines of the present disclosure may beused to induce a balanced immune response against Malaria (e.g., P.falciparum, P. vivax, P. Malariae and/or P. ovale), JapaneseEncephalitis Virus (JEV), West Nile Virus (WNV), Eastern EquineEncephalitis Virus (EEEV), Venezuelan Equine Encephalitis Virus (VEEV),Sindbis Virus (SINV), Chikungunya Virus (CHIKV), Dengue Virus (DENV),Zika Virus (ZIKV) and/or Yellow Fever Virus (YFV), comprising bothcellular and humoral immunity, without risking the possibility ofinsertional mutagenesis, for example. Malaria (e.g., P. falciparum, P.vivax, P. Malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV,DENV, ZIKV and/or YFV are referred to herein as “tropical diseases.”Thus, the terms “tropical disease vaccines” and “Malaria (e.g., P.falciparum, P. vivax, P. Malariae and/or P. ovale), JEV, WNV, EEEV,VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV” encompass Malaria (e.g., P.falciparum, P. vivax, P. Malariae and/or P. ovale) RNA vaccines, JEV RNAvaccines, WNV RNA vaccines, EEEV RNA vaccines, SINV RNA vaccines, CHIKVRNA vaccines, DENV RNA vaccines, ZIKV RNA vaccines, YFV RNA vaccines,and combination vaccines comprising at least two (e.g., at least 3, 4,5, 6, 7, 8 or 9) of any of the Malaria (e.g., P. falciparum, P. vivax,P. Malariae and/or P. ovale) RNA vaccines, JEV RNA vaccines, WNV RNAvaccines, EEEV RNA vaccines, SINV RNA vaccines, CHIKV RNA vaccines, DENVRNA vaccines, ZIKV RNA vaccines, and YFV RNA vaccines.

The RNA (e.g., mRNA) vaccines may be utilized in various settingsdepending on the prevalence of the infection or the degree or level ofunmet medical need. The RNA (e.g. mRNA) vaccines may be utilized totreat and/or prevent Malaria (e.g., P. falciparum, P. vivax, P. Malariaeand/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/orYFV of various genotypes, strains, and isolates. The RNA (e.g., mRNA)vaccines have superior properties in that they produce much largerantibody titers and responses earlier than commercially availableanti-viral therapeutic treatments. While not wishing to be bound bytheory, it is believed that the RNA (e.g., mRNA) vaccines, as mRNApolynucleotides, are better designed to produce the appropriate proteinconformation upon translation, as the RNA (e.g., mRNA) vaccines co-optnatural cellular machinery. Unlike traditional vaccines, which aremanufactured ex vivo and may trigger unwanted cellular responses, RNA(e.g., mRNA) vaccines are presented to the cellular system in a morenative fashion.

Surprisingly, it has been shown that efficacy of mRNA vaccines can besignificantly enhanced when combined with a flagellin adjuvant, inparticular, when one or more antigen-encoding mRNA is combined with anmRNA encoding flagellin. RNA (e.g., mRNA) vaccines combined with theflagellin adjuvant (e.g., mRNA-encoded flagellin adjuvant) have superiorproperties in that they may produce much larger antibody titers andproduce responses earlier than commercially available vaccineformulations.

Some embodiments of the present disclosure provide RNA (e.g., mRNA)vaccines that include at least one RNA (e.g., mRNA) polynucleotidehaving an open reading frame encoding at least one antigenic polypeptideor an immunogenic fragment thereof (e.g., an immunogenic fragmentcapable of inducing an immune response to the antigenic polypeptide) andat least one RNA (e.g., mRNA polynucleotide) having an open readingframe encoding a flagellin adjuvant.

In some embodiments, at least one flagellin polypeptide (e.g., encodedflagellin polypeptide) is a flagellin protein. In some embodiments, atleast one flagellin polypeptide (e.g., encoded flagellin polypeptide) isan immunogenic flagellin fragment. In some embodiments, at least oneflagellin polypeptide and at least one antigenic polypeptide are encodedby a single RNA (e.g., mRNA) polynucleotide. In other embodiments, atleast one flagellin polypeptide and at least one antigenic polypeptideare each encoded by a different RNA polynucleotide.

In some embodiments at least one flagellin polypeptide has at least 80%,at least 85%, at least 90%, or at least 95% identity to a flagellinpolypeptide having a sequence of SEQ ID NO: 420-422.

Provided herein, in some embodiments, is a ribonucleic acid (RNA) (e.g.,mRNA) vaccine, comprising at least one (e.g., at least 2, 3, 4 or 5) RNA(e.g., mRNA) polynucleotide having an open reading frame encoding atleast one (e.g., at least 2, 3, 4 or 5) Malaria (e.g., P. falciparum, P.vivax, P. Malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV,DENV, ZIKV and/or YFV antigenic polypeptide, or any combination of twoor more of the foregoing antigenic polypeptides. Herein, use of the term“antigenic polypeptide” encompasses immunogenic fragments of theantigenic polypeptide (an immunogenic fragment that induces (or iscapable of inducing) an immune response to Malaria (e.g., P. falciparum,P. vivax, P. Malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV,CHIKV, DENV, ZIKV and/or YFV) unless otherwise stated.

Also provided herein, in some embodiments, is a RNA (e.g., mRNA) vaccinecomprising at least one (e.g., at least 2, 3, 4 or 5) RNA polynucleotidehaving an open reading frame encoding at least one (e.g., at least 2, 3,4 or 5) Malaria (e.g., P. falciparum, P. vivax, P. Malariae and/or P.ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFVantigenic polypeptide or an immunogenic fragment thereof, linked to asignal peptide.

Further provided herein, in some embodiments, is a nucleic acid (e.g.,DNA) encoding at least one (e.g., at least 2, 3, 4 or 5) Malaria (e.g.,P. falciparum, P. vivax, P. Malariae and/or P. ovale), JEV, WNV, EEEV,VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV RNA (e.g., mRNA)polynucleotide.

Further still, provided herein, in some embodiments, is a method ofinducing an immune response in a subject, the method comprisingadministering to the subject a vaccine comprising at least one (e.g., atleast 2, 3, 4 or 5) RNA (e.g., mRNA) polynucleotide having an openreading frame encoding at least one (e.g., at least 2, 3, 4 or 5)Malaria (e.g., P. falciparum, P. vivax, P. Malariae and/or P. ovale),JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV antigenicpolypeptide, or any combination of two or more of the foregoingantigenic polypeptides.

Malaria

Some embodiments of the present disclosure provide Malaria vaccines thatinclude at least one ribonucleic acid (RNA) polynucleotide having anopen reading frame encoding at least one Plasmodium (e.g., P.falciparum, P. vivax, P. Malariae and/or P. ovale) antigenic polypeptideor an immunogenic fragment thereof (e.g., an immunogenic fragmentcapable of raising an immune response to Plasmodium).

In some embodiments, the antigenic polypeptide is a circumsporozoite(CS) protein or an immunogenic fragment thereof (e.g., capable ofraising an immune response against Plasmodium).

In some embodiments, the CS protein or fragment is fused to the surfaceantigen from hepatitis B (HBsAg). In some embodiments, the CS protein orfragment is in the form of a hybrid protein comprising substantially allthe C-terminal portion of the CS protein of Plasmodium, four or moretandem repeats of the CS protein immunodominant region, and the surfaceantigen from hepatitis B (HBsAg). In some embodiments, the hybridprotein comprises a sequence of CS protein of Plasmodium falciparumsubstantially as corresponding to amino acids 207-395 of P. falciparumNF54 strain 3D7 clone CS protein fused in frame via a linear linker tothe N-terminal of HBsAg (Ballou W R et al. Am J Trop Med Hyg 2004;71(2_suppl):239-247, incorporated herein by reference).

In some embodiments, the hybrid protein is RTS. In some embodiments, theRTS is in the form of mixed particles RTS,S. In some embodiments, theamount of RTS,S is 25 μg or 50 μg per dose.

In some embodiments, the antigenic polypeptide is liver stage antigen 1(LSA1) or an immunogenic fragment thereof. In some embodiments, theantigenic polypeptide is LSA-NRC.

In some embodiments, the antigenic polypeptide is merozoite surfaceprotein-1 (MSP1) or an immunogenic fragment thereof.

In some embodiments, the antigenic polypeptide is apical membraneantigen 1 (AMA1) or an immunogenic fragment thereof.

In some embodiments, the antigenic polypeptide is thrombospondin relatedadhesive protein (TRAP) or an immunogenic fragment thereof.

In some embodiments, at least one RNA polynucleotide is encoded by atleast one nucleic acid sequence identified by any one of SEQ ID NO: 1-6(Table 1) and homologs having at least 80% identity with a nucleic acidsequence identified by any one of SEQ ID NO: 1-6 (Table 1). In someembodiments, at least one RNA polynucleotide is encoded by at least onenucleic acid sequence identified by any one of SEQ ID NO: 1-6 (Table 1)and homologs having at least 90% (90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.8% or 99.9%) identity with a nucleic acid sequenceidentified by any one of SEQ ID NO: 1-6 (Table 1). In some embodiments,at least one RNA polynucleotide is encoded by at least one fragment of anucleic acid sequence identified by any one of SEQ ID NO: 1-6 (Table 1).

In some embodiments, at least one RNA polynucleotide comprises at leastone nucleic acid sequence identified by any one of SEQ ID NO: 7-12(Table 1) and homologs having at least 80% identity with a nucleic acidsequence identified by any one of SEQ ID NO: 7-12 (Table 1). In someembodiments, at least one RNA polynucleotide comprises at least onenucleic acid sequence identified by any one of SEQ ID NO: 7-12 (Table 1)and homologs having at least 90% (90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.8% or 99.9%) identity with a nucleic acid sequenceidentified by any one of SEQ ID NO: 7-12 (Table 1). In some embodiments,at least one RNA polynucleotide comprises at least one fragment of anucleic acid sequence identified by any one of SEQ ID NO: 7-12 (Table1).

In some embodiments, the at least one RNA polynucleotide encodes atleast one antigenic polypeptide having a sequence identified by any oneof SEQ ID NO: 13-17 (Table 2 and 3). In some embodiments, the at leastone RNA polynucleotide encodes at least one protein variant having atleast 95% identity to an antigenic polypeptide having a sequenceidentified by any one of SEQ ID NO: 13-17 (Table 2 and 3). In someembodiments, at least one antigenic polypeptide has an amino acidsequence identified by any one of SEQ ID NO: 13-17 (Table 2 and 3). Insome embodiments, at least one antigenic polypeptide has at least 95%identity to an antigenic polypeptide having a sequence identified by anyone of SEQ ID NO: 13-17 (Table 2 and 3).

Japanese Encephalitis Virus (JEV)

Some embodiments of the present disclosure provide JEV vaccines thatinclude at least one ribonucleic acid (RNA) polynucleotide having anopen reading frame encoding at least one JEV antigenic polypeptide or animmunogenic fragment thereof (e.g., an immunogenic fragment capable ofinducing an immune response to JEV).

In some embodiments, at least one antigenic polypeptide is JEV Eprotein, JEV Es, JEV prM, JEV capsid, JEV NS1, JEV prM and E polyprotein(prME) or an immunogenic fragment thereof. In some embodiments, at leastone antigenic polypeptide has at least 80%, 85%, 90%, 92%, 95%, 96%,97%, 98% or 99% sequence identity to JEV E protein, JEV Es, JEV prM, JEVcapsid, prME or JEV NS1.

In some embodiments, at least one RNA polynucleotide is encoded by atleast one nucleic acid sequence identified by any one of SEQ ID NO:18-19 (Table 4) and homologs having at least 80% identity with a nucleicacid sequence identified by any one of SEQ ID NO: 18-19 (Table 4). Insome embodiments, at least one RNA polynucleotide is encoded by at leastone nucleic acid sequence identified by any one of SEQ ID NO: 18-19(Table 4) and homologs having at least 90% (90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 99.8% or 99.9%) identity with a nucleic acidsequence identified by any one of SEQ ID NO: 18-19 (Table 4). In someembodiments, at least one RNA polynucleotide is encoded by at least onefragment of a nucleic acid sequence identified by any one of SEQ ID NO:18-19 (Table 4).

In some embodiments, at least one RNA polynucleotide comprises at leastone nucleic acid sequence identified by any one of SEQ ID NO: 20-21(Table 4) and homologs having at least 80% identity with a nucleic acidsequence identified by any one of SEQ ID NO: 20-21 (Table 4). In someembodiments, at least one RNA polynucleotide comprises at least onenucleic acid sequence identified by any one of SEQ ID NO: 20-21 (Table4) and homologs having at least 90% (90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.8% or 99.9%) identity with a nucleic acid sequenceidentified by any one of SEQ ID NO: 20-21 (Table 4). In someembodiments, at least one RNA polynucleotide comprises at least onefragment of a nucleic acid sequence identified by any one of SEQ ID NO:20-21 (Table 4).

In some embodiments, the at least one RNA polynucleotide encodes atleast one antigenic polypeptide having a sequence identified by any oneof SEQ ID NO: 22-29 (Table 5 and 6). In some embodiments, the at leastone RNA polynucleotide encodes at least one protein variant having atleast 95% identity to an antigenic polypeptide having a sequenceidentified by any one of SEQ ID NO: 22-29 (Table 5 and 6). In someembodiments, at least one antigenic polypeptide has an amino acidsequence identified by any one of SEQ ID NO: 22-29 (Table 5 and 6). Insome embodiments, at least one antigenic polypeptide has at least 95%identity to an antigenic polypeptide having a sequence identified by anyone of SEQ ID NO: 22-29 (Table 5 and 6).

West Nile Virus (WNV), Eastern Equine Encephalitis (EEEV), VenezuelanEquine Encephalitis Virus (VEEV), and Sindbis Virus (SINV)

Some embodiments of the present disclosure provide combination vaccinescomprising one or more RNA (e.g., mRNA) polynucleotides. The RNApolynucleotide(s) encode one or more Arbovirus antigens and/or one ormore Alphavirus antigens, on either the same polynucleotide or differentpolynucleotides. RNA polynucleotides featured in the vaccines of thepresent invention can encode one antigen or can encode more than oneantigen, e.g., several antigens (for example, polycistronic RNAs).

In some embodiments, at least one RNA polynucleotide is encoded by atleast one nucleic acid sequence identified by any one of SEQ ID NO:30-34, 48-49, 55-56 (Table 9, 12, 15) and homologs having at least 80%identity with a nucleic acid sequence identified by any one of SEQ IDNO: 30-34, 48-49, 55-56 (Table 9, 12, 15). In some embodiments, at leastone RNA polynucleotide is encoded by at least one nucleic acid sequenceidentified by any one of SEQ ID NO: 30-34, 48-49, 55-56 (Table 9, 12,15) and homologs having at least 90% (90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.8% or 99.9%) identity with a nucleic acid sequenceidentified by any one of SEQ ID NO: 30-34, 48-49, 55-56 (Table 9, 12,15). In some embodiments, at least one RNA polynucleotide is encoded byat least one fragment of a nucleic acid sequence identified by any oneof SEQ ID NO: 30-34, 48-49, 55-56 (Table 9, 12, 15).

In some embodiments, at least one RNA polynucleotide comprises at leastone nucleic acid sequence identified by any one of SEQ ID NO: 35-39,50-51, 57-58 (Table 9, 12, 15) and homologs having at least 80% identitywith a nucleic acid sequence identified by any one of SEQ ID NO: 35-39,50-51, 57-58 (Table 9, 12, 15). In some embodiments, at least one RNApolynucleotide comprises at least one nucleic acid sequence identifiedby any one of SEQ ID NO: 35-39, 50-51, 57-58 (Table 9, 12, 15) andhomologs having at least 90% (90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.8% or 99.9%) identity with a nucleic acid sequenceidentified by any one of SEQ ID NO: 35-39, 50-51, 57-58 (Table 9, 12,15). In some embodiments, at least one RNA polynucleotide comprises atleast one fragment of a nucleic acid sequence identified by any one ofSEQ ID NO: 35-39, 50-51, 57-58 (Table 9, 12, 15).

In some embodiments, the at least one RNA polynucleotide encodes atleast one antigenic polypeptide having a sequence identified by any oneof SEQ ID NO: 44-47, 52-54, 59-64 (Table 10, 11, 13, 14, 16, 17 and 18).In some embodiments, the at least one RNA polynucleotide encodes atleast one protein variant having at least 95% identity to an antigenicpolypeptide having a sequence identified by any one of SEQ ID NO: 44-47,52-54, 59-64 (Table 10, 11, 13, 14, 16, 17 and 18). In some embodiments,at least one antigenic polypeptide has an amino acid sequence identifiedby any one of SEQ ID NO: 44-47, 52-54, 59-64 (Table 10, 11, 13, 14, 16,17 and 18). In some embodiments, at least one antigenic polypeptide hasat least 95% identity to an antigenic polypeptide having a sequenceidentified by any one of SEQ ID NO: 44-47, 52-54, 59-64 (Table 10, 11,13, 14, 16, 17 and 18).

Yellow Fever Virus (YFV)

Yellow fever is an acute viral haemorrhagic disease transmitted byinfected mosquitoes. The “yellow” in the name refers to the jaundicethat affects some patients. Symptoms of yellow fever include fever,headache, jaundice, muscle pain, nausea, vomiting and fatigue. A smallproportion of patients who contract the virus develop severe symptomsand approximately half of those die within 7 to 10 days. Yellow fevervirus (YFV) is endemic in tropical areas of Africa and Central and SouthAmerica. Large epidemics of yellow fever occur when infected peopleintroduce the virus into heavily populated areas with high mosquitodensity and where most people have little or no immunity, due to lack ofvaccination. In these conditions, infected mosquitoes transmit the virusfrom person to person. Since the launch of the Yellow Fever Initiativein 2006, significant progress in combatting the disease has been made inWest Africa and more than 105 million people have been vaccinated inmass campaigns using an attenuated live vaccine. Nonetheless, thisvaccine can cause yellow fever vaccine-associated viscerotropic diseaseas well as yellow fever vaccine-associated neurotropic disease, each ofwhich can be fatal.

Some embodiments of the present disclosure provide Yellow fever virus(YFV) vaccines that include at least one ribonucleic acid (RNA)polynucleotide (e.g., mRNA polynucleotide) having an open reading frameencoding at least one YFV antigenic polypeptide or an immunogenicfragment thereof (e.g., an immunogenic fragment capable of inducing animmune response to YFV).

In some embodiments, the at least one antigenic polypeptide is a YFVpolyprotein.

In some embodiments, the at least one antigenic polypeptide is a YFVcapsid protein, a YFV premembrane/membrane protein, a YFV envelopeprotein, a YFV non-structural protein 1, a YFV non-structural protein2A, a YFV non-structural protein 2B, a YFV non-structural protein 3, aYFV non-structural protein 4A, a YFV non-structural protein 4B, or a YFVnon-structural protein 5.

In some embodiments, the at least one antigenic polypeptide is a YFVcapsid protein or an immunogenic fragment thereof, a YFVpremembrane/membrane protein or an immunogenic fragment thereof, and aYFV envelope protein or an immunogenic fragment thereof.

In some embodiments, the at least one antigenic polypeptide is a YFVcapsid protein or an immunogenic fragment thereof and a YFVpremembrane/membrane protein or an immunogenic fragment thereof.

In some embodiments, the at least one antigenic polypeptide is a YFVcapsid protein or an immunogenic fragment thereof and a YFV envelopeprotein or an immunogenic fragment thereof.

In some embodiments, at least one antigenic polypeptide is a YFVpremembrane/membrane protein or an immunogenic fragment thereof and aYFV envelope protein or an immunogenic fragment thereof.

In some embodiments, the at least one antigenic polypeptide furthercomprises any one or more of a YFV non-structural protein 1, 2A, 2B, 3,4A, 4B or 5.

In some embodiments, at least one RNA polynucleotide is encoded by atleast one nucleic acid sequence identified by any one of SEQ ID NO:65-80 (Table 21) and homologs having at least 80% identity with anucleic acid sequence identified by any one of SEQ ID NO: 65-80 (Table21). In some embodiments, at least one RNA polynucleotide is encoded byat least one nucleic acid sequence identified by any one of SEQ ID NO:65-80 (Table 21) and homologs having at least 90% (90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.8% or 99.9%) identity with a nucleicacid sequence identified by any one of SEQ ID NO: 65-80 (Table 21). Insome embodiments, at least one RNA polynucleotide is encoded by at leastone fragment of a nucleic acid sequence identified by any one of SEQ IDNO: 65-80 (Table 21).

In some embodiments, at least one RNA polynucleotide comprises at leastone nucleic acid sequence identified by any one of SEQ ID NO: 81-96(Table 21) and homologs having at least 80% identity with a nucleic acidsequence identified by any one of SEQ ID NO: 81-96 (Table 21). In someembodiments, at least one RNA polynucleotide comprises at least onenucleic acid sequence identified by any one of SEQ ID NO: 81-96 (Table21) and homologs having at least 90% (90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.8% or 99.9%) identity with a nucleic acid sequenceidentified by any one of SEQ ID NO: 81-96 (Table 21). In someembodiments, at least one RNA polynucleotide comprises at least onefragment of a nucleic acid sequence identified by any one of SEQ ID NO:81-96 (Table 21).

In some embodiments, the at least one RNA polynucleotide encodes atleast one antigenic polypeptide having a sequence identified by any oneof SEQ ID NO: 97-117 (Table 22). In some embodiments, the at least oneRNA polynucleotide encodes at least one protein variant having at least95% identity to an antigenic polypeptide having a sequence identified byany one of SEQ ID NO: 97-117 (Table 22). In some embodiments, at leastone antigenic polypeptide has an amino acid sequence identified by anyone of SEQ ID NO: 97-117 (Table 22). In some embodiments, at least oneantigenic polypeptide has at least 95% identity to an antigenicpolypeptide having a sequence identified by any one of SEQ ID NO: 97-117(Table 22).

Zika Virus (ZIKV)

Zika virus (ZIKV) is a member of the Flaviviridae virus family and theflavivirus genus. In humans, it causes a disease known as Zika fever. Itis related to dengue, yellow fever, West Nile and Japanese encephalitis,viruses that are also members of the virus family Flaviviridae. ZIKV isspread to people through mosquito bites. The most common symptoms ofZIKV disease (Zika) are fever, rash, joint pain, and red eye. Theillness is usually mild with symptoms lasting from several days to aweek. There is no vaccine to prevent, or medicine to treat, Zika virus.

Some embodiments of the present disclosure provide Zika virus (ZIKV)vaccines that include at least one ribonucleic acid (RNA) polynucleotide(e.g., mRNA polynucleotide) having an open reading frame encoding atleast one ZIKV antigenic polypeptide or an immunogenic fragment thereof(e.g., an immunogenic fragment capable of inducing an immune response toZIKV).

In some embodiments, at least one antigenic polypeptide is a ZIKVpolyprotein. In some embodiments, at least one antigenic polypeptide isa ZIKV structural polyprotein. In some embodiments, at least oneantigenic polypeptide is a ZIKV nonstructural polyprotein.

In some embodiments, at least one antigenic polypeptide is a ZIKV capsidprotein, a ZIKV premembrane/membrane protein, a ZIKV envelope protein, aZIKV non-structural protein 1, a ZIKV non-structural protein 2A, a ZIKVnon-structural protein 2B, a ZIKV non-structural protein 3, a ZIKVnon-structural protein 4A, a ZIKV non-structural protein 4B, or a ZIKVnon-structural protein 5.

In some embodiments, at least one antigenic polypeptide is a ZIKV capsidprotein, a ZIKV premembrane/membrane protein, a ZIKV envelope protein, aZIKV non-structural protein 1, a ZIKV non-structural protein 2A, a ZIKVnon-structural protein 2B, a ZIKV non-structural protein 3, a ZIKVnon-structural protein 4A, a ZIKV non-structural protein 4B, or a ZIKVnon-structural protein 5.

In some embodiments, the vaccine comprises a RNA polynucleotide havingan open reading frame encoding a ZIKV capsid protein, a RNApolynucleotide having an open reading frame encoding a ZIKVpremembrane/membrane protein, and a RNA polynucleotide having an openreading frame encoding a ZIKV envelope protein.

In some embodiments, the vaccine comprises a RNA polynucleotide havingan open reading frame encoding a ZIKV capsid protein and a RNApolynucleotide having an open reading frame encoding a ZIKVpremembrane/membrane protein.

In some embodiments, the vaccine comprises a RNA polynucleotide havingan open reading frame encoding a ZIKV capsid protein and a RNApolynucleotide having an open reading frame encoding a ZIKV envelopeprotein.

In some embodiments, the vaccine comprises a RNA polynucleotide havingan open reading frame encoding a ZIKV premembrane/membrane protein and aRNA polynucleotide having an open reading frame encoding a ZIKV envelopeprotein.

In some embodiments, the vaccine comprises a RNA polynucleotide havingan open reading frame encoding a ZIKV capsid protein and at least oneRNA polynucleotide having an open reading frame encoding any one or moreof a ZIKV non-structural protein 1, 2A, 2B, 3, 4A, 4B or 5.

In some embodiments, the vaccine comprises a RNA polynucleotide havingan open reading frame encoding a ZIKV premembrane/membrane protein andat least one RNA polynucleotide having an open reading frame encodingany one or more of a ZIKV non-structural protein 1, 2A, 2B, 3, 4A, 4B or5.

In some embodiments, the vaccine comprises a RNA polynucleotide havingan open reading frame encoding a ZIKV envelope protein and at least oneRNA polynucleotide having an open reading frame encoding any one or moreof a ZIKV non-structural protein 1, 2A, 2B, 3, 4A, 4B or 5.

In some embodiments, the at least one antigenic polypeptide comprises acombination of any two or more of a ZIKV capsid protein, a ZIKVpremembrane/membrane protein, a ZIKV envelope protein, a ZIKVnon-structural protein 1, a ZIKV non-structural protein 2A, a ZIKVnon-structural protein 2B, a ZIKV non-structural protein 3, a ZIKVnon-structural protein 4A, a ZIKV non-structural protein 4B, or a ZIKVnon-structural protein 5.

In some embodiments, at least one RNA polynucleotide is encoded by atleast one nucleic acid sequence identified by any one of SEQ ID NO:118-136 (Table 25) and homologs having at least 80% identity with anucleic acid sequence identified by any one of SEQ ID NO: 118-136 (Table25). In some embodiments, at least one RNA polynucleotide is encoded byat least one nucleic acid sequence identified by any one of SEQ ID NO:118-136 (Table 25) and homologs having at least 90% (90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.8% or 99.9%) identity with a nucleicacid sequence identified by any one of SEQ ID NO: 118-136 (Table 25). Insome embodiments, at least one RNA polynucleotide is encoded by at leastone fragment of a nucleic acid sequence identified by any one of SEQ IDNO: 118-136 (Table 25).

In some embodiments, at least one RNA polynucleotide comprises at leastone nucleic acid sequence identified by any one of SEQ ID NO: 137-155(Table 25) and homologs having at least 80% identity with a nucleic acidsequence identified by any one of SEQ ID NO: 137-155 (Table 25). In someembodiments, at least one RNA polynucleotide comprises at least onenucleic acid sequence identified by any one of SEQ ID NO: 137-155 (Table25) and homologs having at least 90% (90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.8% or 99.9%) identity with a nucleic acid sequenceidentified by any one of SEQ ID NO: 137-155 (Table 25). In someembodiments, at least one RNA polynucleotide comprises at least onefragment of a nucleic acid sequence identified by any one of SEQ ID NO:137-155 (Table 25).

In some embodiments, the at least one RNA polynucleotide encodes atleast one antigenic polypeptide having a sequence identified by any oneof SEQ ID NO: 156-222 or 469 (Table 26 and 27). In some embodiments, theat least one RNA polynucleotide encodes at least one protein varianthaving at least 95% identity to an antigenic polypeptide having asequence identified by any one of SEQ ID NO: 156-222 or 469 (Table 26and 27). In some embodiments, at least one antigenic polypeptide has anamino acid sequence identified by any one of SEQ ID NO: 156-222 or 469(Table 26 and 27). In some embodiments, at least one antigenicpolypeptide has at least 95% identity to an antigenic polypeptide havinga sequence identified by any one of SEQ ID NO: 156-222 or 469 (Table 26and 27).

Dengue Virus (DENV)

Dengue virus (DENV) is a mosquito-borne (Aedes aegypti/Aedes albopictus)member of the family Flaviviridae (positive-sense, single-stranded RNAvirus). Dengue virus is a positive-sense RNA virus of the Flavivirusgenus of the Flaviviridae family, which also includes West Nile virus,Yellow Fever Virus, and Japanese Encephalitis virus. It is transmittedto humans through Stegomyia aegypti (formerly Aedes) mosquito vectorsand is mainly found in the tropical and semitropical areas of the world,where it is endemic in Asia, the Pacific region, Africa, Latin America,and the Caribbean. The incidence of infections has increased 30-foldover the last 50 years (WHO, Dengue: Guidelines for diagnosis,treatment, prevention, and control (2009)) and Dengue virus is thesecond most common tropical infectious disease worldwide after malaria.

Severe disease is most commonly observed in secondary, heterologous DENVinfections. Antibody-dependent enhancement of infection has beenproposed as the primary mechanism of dengue immunopathogenesis. Thepotential risk of immune enhancement of infection and diseaseunderscores the importance of developing dengue vaccines which producebalanced, long-lasting immunity to at least DENV 1-4, if not all five ofthe DENV serotypes. While several dengue vaccines are in development,none have been officially licensed and/or approved to date.

In view of the lack of Dengue virus (DENV) vaccines, there is asignificant need for a vaccine that would be safe and effective in allpatient populations to prevent and/or to treat DENV infection, includingthose individuals at risk for secondary, heterotypic infections (thosewith more than one circulating serotype).

Some embodiments of the present disclosure provide Dengue virus (DENV)vaccines that include at least one ribonucleic acid (RNA) polynucleotide(e.g., mRNA polynucleotide) having an open reading frame encoding atleast one DENV antigenic polypeptide or an immunogenic fragment thereof(e.g., an immunogenic fragment capable of inducing an immune response toDENV).

The methods of the present disclosure, in some embodiments, enable theproduction of highly antigenic DENV RNA vaccines, including RNApolynucleotides encoding concatemeric peptide epitopes. The peptideepitopes are designed to be processed intracellularly and presented tothe immune system in an efficient manner. The RNA (e.g., mRNA) vaccinesdescribed herein are useful for generating a desired immune response byselecting appropriate T or B cell epitopes which are able to bepresented more effectively on MHC-I or MHC-II molecules (depending onwhether they are T or B-cell epitopes, respectively).

In some embodiments, the at least one RNA polynucleotide encodes a DENVcapsid protein or immunogenic fragment or epitope thereof. In someembodiments, the at least one RNA polynucleotide encodes a DENV membraneprotein or immunogenic fragment or epitope thereof. In some embodiments,the at least one RNA polynucleotide encodes a DENV precursor-membraneprotein or immunogenic fragment or epitope thereof. In some embodiments,the at least one RNA polynucleotide encodes a DENV precursor membrane(prM) and envelope (E) polypeptide (DENV prME) or immunogenic fragmentor epitope thereof. In some embodiments, the at least one RNA (e.g.,mRNA) polynucleotide encodes a DENV nonstructural protein or immunogenicfragment or epitope thereof, for example a DENV non-structural proteinselected from NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 proteins, orimmunogenic fragments or epitopes thereof. In some embodiments, the DENVnon-structural protein is NS3.

In some embodiments, the Dengue virus antigen comprises one or moreDengue virus peptide epitopes. In some embodiments, the one or moreDengue virus peptide epitopes is from a DENV envelope protein. In someembodiments, the one or more Dengue virus peptide epitopes is from aDENV capsid protein. In some embodiments, the one or more Dengue viruspeptide epitopes is from a DENV membrane protein. In some embodiments,the one or more Dengue virus peptide epitopes is from a DENVpre-membrane protein. In some embodiments, the one or more Dengue viruspeptide epitopes is from a sequence comprising DENV precursor membrane(prM) and envelope (E) polypeptide (DENV prME). In some embodiments, theat least one Dengue virus antigen is a DENV2 prME peptide epitope. Insome embodiments, the one or more Dengue virus peptide epitopes is froma DENV nonstructural protein.

In any of these embodiments, the at least one RNA (e.g., mRNA)polynucleotide encodes a DENV polypeptide, fragment, or epitope from aDENV serotype selected from DENV-1, DENV-2, DENV-3, DENV-4, and DENV-5.In some embodiments, the one or more Dengue virus peptide epitopes isfrom a DENV-2 serotype. In some embodiments, the one or more Denguevirus peptide epitopes is from a DENV2 membrane polypeptide. In someembodiments, the one or more Dengue virus peptide epitopes is from aDENV2 envelope polypeptide. In some embodiments, the one or more Denguevirus peptide epitopes is from a DENV2 pre-membrane polypeptide. In someembodiments, the one or more Dengue virus peptide epitopes is from aDENV2 capsid polypeptide. In some embodiments, the one or more Denguevirus peptide epitopes is from a DENV2 non-structural polypeptide. Insome embodiments, the one or more Dengue virus peptide epitopes is froma DENV2 pre-membrane polypeptide. In some embodiments, the one or moreDengue virus peptide epitopes is from a DENV2 PrME polypeptide.

In some embodiments, the Dengue virus antigen is a concatemeric Denguevirus antigen comprising two or more Dengue virus peptide epitopes. Insome embodiments, the Dengue virus concatemeric antigen comprisesbetween 2-100 Dengue peptide epitopes interspersed by cleavage sensitivesites. In some embodiments, the peptide epitopes are not epitopes ofantibody dependent enhancement. In some embodiments, the Dengue virusvaccine's peptide epitopes are T cell epitopes and/or B cell epitopes.In other embodiments, the Dengue virus vaccine's peptide epitopescomprise a combination of T cell epitopes and B cell epitopes. In someembodiments, at least one of the peptide epitopes of the Dengue virusvaccine is a T cell epitope.

In some embodiments, the protease cleavage site of the Dengue virusvaccine comprises the amino acid sequence GFLG (SEQ ID NO: 429), KVSR(SEQ ID NO: 430), TVGLR (SEQ ID NO: 431), PMGLP (SEQ ID NO: 432), orPMGAP (SEQ ID NO: 433).

In some embodiments, the at least one RNA polynucleotide encodes a DENVenvelope protein, and one or more concatemeric Dengue virus antigen(s),such as any of the concatemeric antigens described herein. In someembodiments, the at least one RNA polynucleotide encodes a DENV membraneprotein, and a concatemeric virus antigen, such as any of theconcatemeric antigens described herein. In some embodiments, the atleast one RNA polynucleotide encodes a DENV capsid protein and aconcatemeric virus antigen, such as any of the concatemeric antigensdescribed herein. In some embodiments, the at least one RNApolynucleotide encodes a DENV nonstructural protein, for example a DENVnon-structural protein selected from NS1, NS2A, NS2B, NS3, SN4A, NS4B,and NS5 proteins, and a concatemeric Dengue virus antigen, such as anyof the concatemeric antigens described herein. In some embodiments, theDENV non-structural protein is NS3. In some embodiments, the at leastone RNA polynucleotide encodes a DENV precursor membrane protein, andone or more concatemeric Dengue virus antigen(s), such as any of theconcatemeric antigens described herein. In some embodiments, the atleast one RNA polynucleotide encodes a DENV prME polypeptide, and one ormore concatemeric Dengue virus antigen(s), such as any of theconcatemeric antigens described herein.

In some embodiments, the peptide epitopes comprise at least one MHCclass I epitope and at least one MHC class II epitope. In someembodiments, at least 10% of the epitopes are MHC class I epitopes. Insome embodiments, at least 20% of the epitopes are MHC class I epitopes.In some embodiments, at least 30% of the epitopes are MHC class Iepitopes. In some embodiments, at least 40% of the epitopes are MHCclass I epitopes. In some embodiments, at least 50%, 60%, 70%, 80%, 90%or 100% of the epitopes are MHC class I epitopes. In some embodiments,at least 10% of the epitopes are MHC class II epitopes. In someembodiments, at least 20% of the epitopes are MHC class II epitopes. Insome embodiments, at least 30% of the epitopes are MHC class IIepitopes. In some embodiments, at least 40% of the epitopes are MHCclass II epitopes. In some embodiments, at least 50%, 60%, 70%, 80%, 90%or 100% of the epitopes are MHC class II epitopes. In some embodiments,the ratio of MHC class I epitopes to MHC class II epitopes is a ratioselected from about 10%:about 90%;about 20%:about 80%;about 30%:about70%;about 40%:about 60%;about 50%:about 50%;about 60%:about 40%;about70%:about 30%;about 80%:about 20%;about 90%:about 10% MHC class 1:MHCclass II epitopes. In some embodiments, the ratio of MHC class IIepitopes to MHC class I epitopes is a ratio selected from about10%:about 90%;about 20%:about 80%;about 30%:about 70%;about 40%:about60%;about 50%:about 50%;about 60%:about 40%;about 70%:about 30%;about80%:about 20%;about 90%:about 10% MHC class I1:MHC class I epitopes. Insome embodiments, at least one of the peptide epitopes of the Denguevirus vaccine is a B cell epitope. In some embodiments, the T cellepitope of the Dengue virus vaccine comprises between 8-11 amino acids.In some embodiments, the B cell epitope of the Dengue virus vaccinecomprises between 13-17 amino acids.

In any of these embodiments, the concatemeric Dengue virus antigen maycomprise two or more Dengue virus peptide epitopes selected from a DENVenvelope polypeptide, DENV capsid polypeptide, DENV membranepolypeptide, DENV precursor-membrane polypeptide, DENV nonstructuralpolypeptide, DENV prME polypeptide, and any combination thereof, and thetwo or more Dengue virus peptide epitopes may be from any DENV serotype,for example, a DENV serotype selected from DENV-1, DENV-2, DENV-3,DENV-4, DENV-5 and combinations thereof. In some embodiments, theconcatemeric Dengue virus antigen comprises two or more Dengue viruspeptide epitopes from DENV-2 serotype. In some embodiments, theconcatemeric Dengue virus antigen comprises two or more DENV2 prMEpeptide epitopes, which may be the same or different DENV prME peptideepitopes.

In some embodiments, at least one RNA polynucleotide is encoded by atleast one nucleic acid sequence identified by any one of SEQ ID NO:223-239 (Table 28) and homologs having at least 80% identity with anucleic acid sequence identified by any one of SEQ ID NO: 223-239 (Table28). In some embodiments, at least one RNA polynucleotide is encoded byat least one nucleic acid sequence identified by any one of SEQ ID NO:223-239 (Table 28) and homologs having at least 90% (90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.8% or 99.9%) identity with a nucleicacid sequence identified by any one of SEQ ID NO: 223-239 (Table 28). Insome embodiments, at least one RNA polynucleotide is encoded by at leastone fragment of a nucleic acid sequence identified by any one of SEQ IDNO: 223-239 (Table 28).

In some embodiments, at least one RNA polynucleotide comprises at leastone nucleic acid sequence identified by any one of SEQ ID NO: 240-256(Table 28) and homologs having at least 80% identity with a nucleic acidsequence identified by any one of SEQ ID NO: 240-256 (Table 28). In someembodiments, at least one RNA polynucleotide comprises at least onenucleic acid sequence identified by any one of SEQ ID NO: 240-256 (Table28) and homologs having at least 90% (90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.8% or 99.9%) identity with a nucleic acid sequenceidentified by any one of SEQ ID NO: 240-256 (Table 28). In someembodiments, at least one RNA polynucleotide comprises at least onefragment of a nucleic acid sequence identified by any one of SEQ ID NO:240-256 (Table 28).

In some embodiments, the at least one RNA polynucleotide encodes atleast one antigenic polypeptide having a sequence identified by any oneof SEQ ID NO: 259-291 (Table 29 and 42). In some embodiments, the atleast one RNA polynucleotide encodes at least one protein variant havingat least 95% identity to an antigenic polypeptide having a sequenceidentified by any one of SEQ ID NO: 259-291 (Table 29 and 42). In someembodiments, at least one antigenic polypeptide has an amino acidsequence identified by any one of SEQ ID NO: 259-291 (Table 29 and 42).In some embodiments, at least one antigenic polypeptide has at least 95%identity to an antigenic polypeptide having a sequence identified by anyone of SEQ ID NO: 259-291 (Table 29 and 42).

Chikungunya Virus (CHIKV)

Some embodiments of the present disclosure provide Chikungunya virus(CHIKV) vaccines that include at least one ribonucleic acid (RNA)polynucleotide (e.g., mRNA polynucleotide) having an open reading frameencoding at least one CHIKV antigenic polypeptide or an immunogenicfragment thereof (e.g., an immunogenic fragment capable of inducing animmune response to CHIKV).

Chikungunya virus (CHIKV) is a mosquito-borne virus belonging to theAlphavirus genus of the Togaviridae family that was first isolated in1953 in Tanzania, where the virus was endemic. Outbreaks occurrepeatedly in west, central, and southern Africa and have caused severalhuman epidemics in those areas since that time. The virus is passed tohumans by two species of mosquito of the genus Aedes: A. albopictus andA. aegypti. There are several Chikungunya genotypes: Indian Ocean,East/Central/South African (ECSA), Asian, West African, and Brazilian.

The CHIKV antigenic polypeptide may be a Chikungunya structural proteinor an antigenic fragment or epitope thereof. In some embodiments, theantigenic polypeptide is a CHIKV structural protein or an antigenicfragment thereof. For example, a CHIKV structural protein may be anenvelope protein (E), a 6K protein, or a capsid (C) protein. In someembodiments, the CHIKV structural protein is an envelope proteinselected from E1, E2, and E3. In some embodiments, the CHIKV structuralprotein is E1 or E2. In some embodiments, the CHIKV structural proteinis a capsid protein. In some embodiments, the antigenic polypeptide is afragment or epitope of a CHIKV structural protein.

In some embodiments, the antigenic polypeptide comprises two or moreCHIKV structural proteins. In some embodiments, the two or more CHIKVstructural proteins are envelope proteins. In some embodiments, the twoor more CHIKV structural proteins are E1 and E2. In some embodiments,the two or more CHIKV structural proteins are E1 and E3. In someembodiments, the two or more CHIKV structural proteins are E2 and E3. Insome embodiments, the two or more CHIKV structural proteins are E1, E2,and E3. In some embodiments, the two or more CHIKV structural proteinsare envelope and capsid proteins. In some embodiments, the two or moreCHIKV structural proteins are E1 and C. In some embodiments, the two ormore CHIKV structural proteins are E2 and C. In some embodiments, thetwo or more CHIKV structural proteins are E3 and C. In some embodiments,the two or more CHIKV structural proteins are E1, E2, and C. In someembodiments, the two or more CHIKV structural proteins are E1, E3, andC. In some embodiments, the two or more CHIKV structural proteins areE2, E3, and C. In some embodiments, the two or more CHIKV structuralproteins are E1, E2, E3, and C. In some embodiments, the two or moreCHIKV structural proteins are E1, 6K, and E2. In some embodiments, thetwo or more CHIKV structural proteins are E2, 6K, and E3. In someembodiments, the two or more CHIKV structural proteins are E1, 6K, andE3. In some embodiments, the two or more CHIKV structural proteins areE1, E2, E3, 6K, and C. In some embodiments, the antigenic polypeptidecomprises the CHIKV structural polyprotein comprising C, E3, E2, 6K, andE1. In some embodiments, the antigenic polypeptide is a fragment orepitope of two or more CHIKV structural proteins or a fragment orepitope of the polyprotein.

In some embodiments, at least one RNA polynucleotide is encoded by atleast one nucleic acid sequence identified by any one of SEQ ID NO:376-388 (Table 47) and homologs having at least 80% identity with anucleic acid sequence identified by any one of SEQ ID NO: 376-388 (Table47). In some embodiments, at least one RNA polynucleotide is encoded byat least one nucleic acid sequence identified by any one of SEQ ID NO:376-388 (Table 47) and homologs having at least 90% (90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.8% or 99.9%) identity with a nucleicacid sequence identified by any one of SEQ ID NO: 376-388 (Table 47). Insome embodiments, at least one RNA polynucleotide is encoded by at leastone fragment of a nucleic acid sequence identified by any one of SEQ IDNO: 376-388 (Table 47).

In some embodiments, at least one RNA polynucleotide comprises at leastone nucleic acid sequence identified by any one of SEQ ID NO: 389-401(Table 47) and homologs having at least 80% identity with a nucleic acidsequence identified by any one of SEQ ID NO: 389-401 (Table 47). In someembodiments, at least one RNA polynucleotide comprises at least onenucleic acid sequence identified by any one of SEQ ID NO: 389-401 (Table47) and homologs having at least 90% (90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.8% or 99.9%) identity with a nucleic acid sequenceidentified by any one of SEQ ID NO: 389-401 (Table 47). In someembodiments, at least one RNA polynucleotide comprises at least onefragment of a nucleic acid sequence identified by any one of SEQ ID NO:389-401 (Table 47).

In some embodiments, the at least one RNA polynucleotide encodes atleast one antigenic polypeptide having a sequence identified by any oneof SEQ ID NO: 402-413 (Table 48). In some embodiments, the at least oneRNA polynucleotide encodes at least one protein variant having at least95% identity to an antigenic polypeptide having a sequence identified byany one of SEQ ID NO: 402-413 (Table 48). In some embodiments, at leastone antigenic polypeptide has an amino acid sequence identified by anyone of SEQ ID NO: 402-413 (Table 48). In some embodiments, at least oneantigenic polypeptide has at least 95% identity to an antigenicpolypeptide having a sequence identified by any one of SEQ ID NO:402-413 (Table 48).

In some embodiments, an open reading frame of a RNA (e.g., mRNA) vaccineis codon-optimized. In some embodiments, at least one RNA polynucleotideencodes at least one antigenic polypeptide comprising an amino acidsequence identified by any one of SEQ ID NO: 13-17, 22-29, 44-47, 52-54,59-64, 97-117, 156-222, 469, 259-291 or 402-413 and is codon optimizedmRNA.

In some embodiments, a RNA (e.g., mRNA) vaccine further comprising anadjuvant.

Tables 3, 6, 11, 14, 17, 27, and 42 provide National Center forBiotechnology Information (NCBI) accession numbers of interest. Itshould be understood that the phrase “an amino acid sequence of Tables3, 6, 11, 14, 17, 27, and 42” refers to an amino acid sequenceidentified by one or more NCBI accession numbers listed in Tables 3, 6,11, 14, 17, 27, and 42. Each of the amino acid sequences, and variantshaving greater than 95% identity or greater than 98% identity to each ofthe amino acid sequences encompassed by the accession numbers of Tables3, 6, 11, 14, 17, 27, and 42 are included within the constructs(polynucleotides/polypeptides) of the present disclosure.

In some embodiments, at least one mRNA polynucleotide is encoded by anucleic acid comprising a sequence identified by any one of SEQ ID NO:1-6, 18, 19, 30-34, 48, 49, 55, 56, 65-80, 118-136, 223-239 or 376-388and having less than 80% identity to wild-type mRNA sequence. In someembodiments, at least one mRNA polynucleotide is encoded by a nucleicacid comprising a sequence identified by any one of SEQ ID NO: 1-6, 18,19, 30-34, 48, 49, 55, 56, 65-80, 118-136, 223-239 or 376-388 and havingless than 75%, 85% or 95% identity to a wild-type mRNA sequence. In someembodiments, at least one mRNA polynucleotide is encoded by nucleic acidcomprising a sequence identified by any one of SEQ ID NO: 1-6, 18, 19,30-34, 48, 49, 55, 56, 65-80, 118-136, 223-239 or 376-388 and havingless than 50-80%, 60-80%, 40-80%, 30-80%, 70-80%, 75-80% or 78-80%identity to wild-type mRNA sequence. In some embodiments, at least onemRNA polynucleotide is encoded by a nucleic acid comprising a sequenceidentified by any one of SEQ ID NO: 1-6, 18, 19, 30-34, 48, 49, 55, 56,65-80, 118-136, 223-239 or 376-388 and having less than 40-85%, 50-85%,60-85%, 30-85%, 70-85%, 75-85% or 80-85% identity to wild-type mRNAsequence. In some embodiments, at least one mRNA polynucleotide isencoded by a nucleic acid comprising a sequence identified by any one ofSEQ ID NO: 1-6, 18, 19, 30-34, 48, 49, 55, 56, 65-80, 118-136, 223-239or 376-388 and having less than 40-90%, 50-90%, 60-90%, 30-90%, 70-90%,75-90%, 80-90%, or 85-90% identity to wild-type mRNA sequence.

In some embodiments, at least one mRNA polynucleotide comprises anucleic acid comprising a sequence identified by any one of SEQ ID NO:7-12, 20-21, 35-39, 50-51, 57-58, 81-96, 137-155, 240-256, or 389-401(with or without a signal sequence, 5′ UTR, 3′ UTR, and/or polyA tail)and having less than 80% identity to wild-type mRNA sequence. In someembodiments, at least one mRNA polynucleotide comprises a nucleic acidcomprising a sequence identified by any one of SEQ ID NO: 7-12, 20-21,35-39, 50-51, 57-58, 81-96, 137-155, 240-256, or 389-401 (with orwithout a signal sequence, 5′ UTR, 3′ UTR, and/or polyA tail) and havingless than 75%, 85% or 95% identity to a wild-type mRNA sequence. In someembodiments, at least one mRNA polynucleotide comprises nucleic acidcomprising a sequence identified by any one of SEQ ID NO: 7-12, 20-21,35-39, 50-51, 57-58, 81-96, 137-155, 240-256, or 389-401 (with orwithout a signal sequence, 5′ UTR, 3′ UTR, and/or polyA tail) and havingless than 50-80%, 60-80%, 40-80%, 30-80%, 70-80%, 75-80% or 78-80%identity to wild-type mRNA sequence. In some embodiments, at least onemRNA polynucleotide comprises a nucleic acid comprising a sequenceidentified by any one of SEQ ID NO: 7-12, 20-21, 35-39, 50-51, 57-58,81-96, 137-155, 240-256, or 389-401 (with or without a signal sequence,5′ UTR, 3′ UTR, and/or polyA tail) and having less than 40-85%, 50-85%,60-85%, 30-85%, 70-85%, 75-85% or 80-85% identity to wild-type mRNAsequence. In some embodiments, at least one mRNA polynucleotidecomprises a nucleic acid comprising a sequence identified by any one ofSEQ ID NO: 7-12, 20-21, 35-39, 50-51, 57-58, 81-96, 137-155, 240-256, or389-401 (with or without a signal sequence, 5′ UTR, 3′ UTR, and/or polyAtail) and having less than 40-90%, 50-90%, 60-90%, 30-90%, 70-90%,75-90%, 80-90%, or 85-90% identity to wild-type mRNA sequence.

In some embodiments, at least one RNA polynucleotide encodes at leastone antigenic polypeptide comprising an amino acid sequence identifiedby any one of SEQ ID NO: 13-17, 22-29, 44-47, 52-54, 59-64, 97-117,156-222, 469, 259-291 or 402-413 and having at least 80% (e.g., 85%,90%, 95%, 98%, 99%) identity to wild-type mRNA sequence, but does notinclude wild-type mRNA sequence.

In some embodiments, at least one RNA polynucleotide encodes at leastone antigenic polypeptide comprising an amino acid sequence identifiedby any one of SEQ ID NO: 13-17, 22-29, 44-47, 52-54, 59-64, 97-117,156-222, 469, 259-291 or 402-413 and has less than 95%, 90%, 85%, 80% or75% identity to wild-type mRNA sequence. In some embodiments, at leastone RNA polynucleotide encodes at least one antigenic polypeptidecomprising an amino acid sequence identified by any one of SEQ ID NO:13-17, 22-29, 44-47, 52-54, 59-64, 97-117, 156-222, 469, 259-291 or402-413 and has 30-80%, 40-80%, 50-80%, 60-80%, 70-80%, 75-80% or78-80%, 30-85%, 40-85%, 50-85%, 60-85%, 70-85%, 75-85% or 78-85%,30-90%, 40-90%, 50-90%, 60-90%, 70-90%, 75-90%, 80-90% or 85-90%identity to wild-type mRNA sequence.

In some embodiments, at least one RNA polynucleotide encodes at leastone antigenic polypeptide having at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identity to an aminoacid sequence identified by any one of SEQ ID NO: 13-17, 22-29, 44-47,52-54, 59-64, 97-117, 156-222, 469, 259-291 or 402-413. In someembodiments, at least one RNA polynucleotide encodes at least oneantigenic polypeptide having 95-99% identity to an amino acid sequenceidentified by any one of SEQ ID NO: 13-17, 22-29, 44-47, 52-54, 59-64,97-117, 156-222, 469, 259-291 or 402-413.

In some embodiments, at least one RNA polynucleotide encodes at leastone antigenic polypeptide having at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identity to amino acidsequence identified by any one of SEQ ID NO: 13-17, 22-29, 44-47, 52-54,59-64, 97-117, 156-222, 469, 259-291 or 402-413 and having membranefusion activity. In some embodiments, at least one RNA polynucleotideencodes at least one antigenic polypeptide having 95-99% identity toamino acid sequence identified by any one of SEQ ID NO: 13-17, 22-29,44-47, 52-54, 59-64, 97-117, 156-222, 469, 259-291 or 402-413 and havingmembrane fusion activity.

In some embodiments, at least one RNA polynucleotide encodes at leastone antigenic polypeptide (e.g., at least one Malaria (e.g., P.falciparum, P. vivax, P. Malariae and/or P. ovale), JEV, WNV, EEEV,VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV antigenic polypeptide) thatattaches to cell receptors.

In some embodiments, at least one RNA polynucleotide encodes at leastone antigenic polypeptide (e.g., at least one Malaria (e.g., P.falciparum, P. vivax, P. Malariae and/or P. ovale), JEV, WNV, EEEV,VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV antigenic polypeptide)antigenic polypeptide) that causes fusion of viral and cellularmembranes.

In some embodiments, at least one RNA polynucleotide encodes at leastone antigenic polypeptide (e.g., at least one Malaria (e.g., P.falciparum, P. vivax, P. Malariae and/or P. ovale), JEV, WNV, EEEV,VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV antigenic polypeptide) that isresponsible for binding of the virus to a cell being infected.

Some embodiments of the present disclosure provide a vaccine thatincludes at least one ribonucleic acid (RNA) (e.g., mRNA) polynucleotidehaving an open reading frame encoding at least one antigenic polypeptide(e.g., at least one Malaria (e.g., P. falciparum, P. vivax, P. Malariaeand/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/orYFV antigenic polypeptide), at least one 5′ terminal cap and at leastone chemical modification, formulated within a lipid nanoparticle.

In some embodiments, a 5′ terminal cap is 7 mG(5′)ppp(5′)NlmpNp.

In some embodiments, at least one chemical modification is selected frompseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine,2-thiouridine, 4′-thiouridine, 5-methylcytosine, 5-methyluridine,2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine,2-thio-5-aza-uridine, 2-thio-dihydropseudouridine,2-thio-dihydrouridine, 2-thio-pseudouridine,4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine,4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine,dihydropseudouridine, 5-methoxyuridine and 2′-O-methyl uridine. In someembodiments, the chemical modification is in the 5-position of theuracil. In some embodiments, the chemical modification is aN1-methylpseudouridine or a N1-ethylpseudouridine. In some embodiments,a lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, asterol and a non-cationic lipid. In some embodiments, a cationic lipidis an ionizable cationic lipid and the non-cationic lipid is a neutrallipid, and the sterol is a cholesterol. In some embodiments, a cationiclipid is selected from the group consisting of2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA),di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319),(12Z,15Z)—N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine (L608), andN,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecan-8-amine (L530).

In some embodiments, the lipid is

In some embodiments, the lipid is

In some embodiments, a lipid nanoparticle comprises compounds of Formula(I) and/or Formula (II), discussed below.

In some embodiments, a lipid nanoparticle comprises Compounds 3, 18, 20,25, 26, 29, 30, 60, 108-112, or 122, as discussed below.

Some embodiments of the present disclosure provide a vaccine thatincludes at least one RNA (e.g., mRNA) polynucleotide having an openreading frame encoding at least one antigenic polypeptide (e.g., atleast one Malaria (e.g., P. falciparum, P. vivax, P. Malariae and/or P.ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFVantigenic polypeptide), wherein at least 80% (e.g., 85%, 90%, 95%, 98%,99%) of the uracil in the open reading frame have a chemicalmodification, optionally wherein the vaccine is formulated in a lipidnanoparticle (e.g., a lipid nanoparticle comprises a cationic lipid, aPEG-modified lipid, a sterol and a non-cationic lipid).

In some embodiments, 100% of the uracil in the open reading frame have achemical modification. In some embodiments, a chemical modification isin the 5-position of the uracil. In some embodiments, a chemicalmodification is a N1-methyl pseudouridine. In some embodiments, 100% ofthe uracil in the open reading frame have a N1-methyl pseudouridine inthe 5-position of the uracil.

In some embodiments, an open reading frame of a RNA (e.g., mRNA)polynucleotide encodes at least two antigenic polypeptides (e.g., atleast one Malaria (e.g., P. falciparum, P. vivax, P. Malariae and/or P.ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFVantigenic polypeptide). In some embodiments, the open reading frameencodes at least five or at least ten antigenic polypeptides. In someembodiments, the open reading frame encodes at least 100 antigenicpolypeptides. In some embodiments, the open reading frame encodes 2-100antigenic polypeptides.

In some embodiments, a vaccine comprises at least two RNA (e.g., mRNA)polynucleotides, each having an open reading frame encoding at least oneantigenic polypeptide (e.g., at least one Malaria (e.g., P. falciparum,P. vivax, P. Malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV,CHIKV, DENV, ZIKV and/or YFV antigenic polypeptide). In someembodiments, the vaccine comprises at least five or at least ten RNA(e.g., mRNA) polynucleotides, each having an open reading frame encodingat least one antigenic polypeptide or an immunogenic fragment thereof.In some embodiments, the vaccine comprises at least 100 RNA (e.g., mRNA)polynucleotides, each having an open reading frame encoding at least oneantigenic polypeptide. In some embodiments, the vaccine comprises 2-100RNA (e.g., mRNA) polynucleotides, each having an open reading frameencoding at least one antigenic polypeptide.

In some embodiments, at least one antigenic polypeptide (e.g., at leastone Malaria (e.g., P. falciparum, P. vivax, P. Malariae and/or P.ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFVantigenic polypeptide) is fused to a signal peptide. In someembodiments, the signal peptide is selected from: a HuIgGk signalpeptide (METPAQLLFLLLLWLPDTTG; SEQ ID NO: 423); IgE heavy chainepsilon-1 signal peptide (MDWTWILFLVAAATRVHS; SEQ ID NO: 424); Japaneseencephalitis PRM signal sequence (MLGSNSGQRVVFTILLLLVAPAYS; SEQ ID NO:425), VSINVg protein signal sequence (MKCLLYLAFLFIGVNCA; SEQ ID NO: 426)and Japanese encephalitis JEV signal sequence (MWLVSLAIVTACAGA; SEQ IDNO: 427).

In some embodiments, the signal peptide is fused to the N-terminus of atleast one antigenic polypeptide. In some embodiments, a signal peptideis fused to the C-terminus of at least one antigenic polypeptide.

In some embodiments, at least one antigenic polypeptide (e.g., at leastone Malaria (e.g., P. falciparum, P. vivax, P. Malariae and/or P.ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFVantigenic polypeptide) comprises a mutated N-linked glycosylation site.

Also provided herein is a RNA (e.g., mRNA) vaccine of any one of theforegoing paragraphs (e.g., at least one Malaria (e.g., P. falciparum,P. vivax, P. Malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV,CHIKV, DENV, ZIKV and/or YFV antigenic polypeptide), formulated in ananoparticle (e.g., a lipid nanoparticle).

In some embodiments, the nanoparticle has a mean diameter of 50-200 nm.In some embodiments, the nanoparticle is a lipid nanoparticle.

In some embodiments, a lipid nanoparticle comprises compounds of Formula(I) and/or Formula (II), discussed below.

In some embodiments, a tropical disease RNA (e.g., mRNA) vaccine isformulated in a lipid nanoparticle that comprises a compound selectedfrom Compounds 3, 18, 20, 25, 26, 29, 30, 60, 108-112 and 122, describedbelow.

In some embodiments, the nanoparticle has a polydispersity value of lessthan 0.4 (e.g., less than 0.3, 0.2 or 0.1).

In some embodiments, the nanoparticle has a net neutral charge at aneutral pH value.

In some embodiments, the RNA (e.g., mRNA) vaccine is multivalent.

Some embodiments of the present disclosure provide methods of inducingan antigen specific immune response in a subject, comprisingadministering to the subject any of the RNA (e.g., mRNA) vaccine asprovided herein in an amount effective to produce an antigen-specificimmune response. In some embodiments, the RNA (e.g., mRNA) vaccine is aMalaria (e.g., P. falciparum, P. vivax, P. Malariae and/or P. ovale),JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV vaccine. Insome embodiments, the RNA (e.g., mRNA) vaccine is a combination vaccinecomprising a combination of Malaria (e.g., P. falciparum, P. vivax, P.Malariae and/or P. ovale) vaccine, JEV vaccine, WNV vaccine, EEEVvaccine, SINV vaccine, CHIKV vaccine, DENV vaccine, ZIKV vaccine and/orYFV vaccine.

In some embodiments, an antigen-specific immune response comprises a Tcell response or a B cell response.

In some embodiments, a method of producing an antigen-specific immuneresponse comprises administering to a subject a single dose (no boosterdose) of a RNA (e.g., mRNA) vaccine of the present disclosure. In someembodiments, the RNA (e.g., mRNA) vaccine is a Malaria (e.g., P.falciparum, P. vivax, P. Malariae and/or P. ovale) vaccine, JEV vaccine,WNV vaccine, EEEV vaccine, SINV vaccine, CHIKV vaccine, DENV vaccine,ZIKV vaccine and/or YFV vaccine. In some embodiments, the RNA (e.g.,mRNA) vaccine is a combination vaccine comprising a combination of anytwo or more of the foregoing vaccines.

In some embodiments, a method further comprises administering to thesubject a second (booster) dose of a RNA (e.g., mRNA) vaccine.Additional doses of a RNA (e.g., mRNA) vaccine may be administered.

In some embodiments, the subjects exhibit a seroconversion rate of atleast 80% (e.g., at least 85%, at least 90%, or at least 95%) followingthe first dose or the second (booster) dose of the vaccine.Seroconversion is the time period during which a specific antibodydevelops and becomes detectable in the blood. After seroconversion hasoccurred, a virus can be detected in blood tests for the antibody.During an infection or immunization, antigens enter the blood, and theimmune system begins to produce antibodies in response. Beforeseroconversion, the antigen itself may or may not be detectable, butantibodies are considered absent. During seroconversion, antibodies arepresent but not yet detectable. Any time after seroconversion, theantibodies can be detected in the blood, indicating a prior or currentinfection.

In some embodiments, a RNA (e.g., mRNA) vaccine is administered to asubject by intradermal, intramuscular injection, or by intranasaladministration.

Some embodiments of the present disclosure provide methods of inducingan antigen specific immune response in a subject, includingadministering to a subject a RNA (e.g., mRNA) vaccine in an effectiveamount to produce an antigen specific immune response in a subject.Antigen-specific immune responses in a subject may be determined, insome embodiments, by assaying for antibody titer (for titer of anantibody that binds to a Malaria (e.g., P. falciparum, P. vivax, P.Malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKVand/or YFV antigenic polypeptide) following administration to thesubject of any of the RNA (e.g., mRNA) vaccines of the presentdisclosure. In some embodiments, the anti-antigenic polypeptide antibodytiter produced in the subject is increased by at least 1 log relative toa control. In some embodiments, the anti-antigenic polypeptide antibodytiter produced in the subject is increased by 1-3 log relative to acontrol.

In some embodiments, the anti-antigenic polypeptide antibody titerproduced in a subject is increased at least 2 times relative to acontrol. In some embodiments, the anti-antigenic polypeptide antibodytiter produced in the subject is increased at least 5 times relative toa control. In some embodiments, the anti-antigenic polypeptide antibodytiter produced in the subject is increased at least 10 times relative toa control. In some embodiments, the anti-antigenic polypeptide antibodytiter produced in the subject is increased 2-10 times relative to acontrol.

In some embodiments, the control is an anti-antigenic polypeptideantibody titer produced in a subject who has not been administered a RNA(e.g., mRNA) vaccine of the present disclosure. In some embodiments, thecontrol is an anti-antigenic polypeptide antibody titer produced in asubject who has been administered a live attenuated or inactivatedMalaria (e.g., P. falciparum, P. vivax, P. Malariae and/or P. ovale),JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV vaccine (see,e.g., Ren J. et al. J of Gen. Virol. 2015; 96: 1515-1520), or whereinthe control is an anti-antigenic polypeptide antibody titer produced ina subject who has been administered a recombinant or purified Malaria(e.g., P. falciparum, P. vivax, P. Malariae and/or P. ovale), JEV, WNV,EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV protein vaccine. In someembodiments, the control is an anti-antigenic polypeptide antibody titerproduced in a subject who has been administered a Malaria (e.g., P.falciparum, P. vivax, P. Malariae and/or P. ovale), JEV, WNV, EEEV,VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV virus-like particle (VLP)vaccine (see, e.g., Cox R G et al., J Virol. 2014 June; 88(11):6368-6379).

A RNA (e.g., mRNA) vaccine of the present disclosure is administered toa subject in an effective amount (an amount effective to induce animmune response). In some embodiments, the effective amount is a doseequivalent to an at least 2-fold, at least 4-fold, at least 10-fold, atleast 100-fold, at least 1000-fold reduction in the standard of caredose of a recombinant Malaria (e.g., P. falciparum, P. vivax, P.Malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKVand/or YFV protein vaccine, wherein the anti-antigenic polypeptideantibody titer produced in the subject is equivalent to ananti-antigenic polypeptide antibody titer produced in a control subjectadministered the standard of care dose of a recombinant Malaria (e.g.,P. falciparum, P. vivax, P. Malariae and/or P. ovale), JEV, WNV, EEEV,VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV protein vaccine, a purifiedMalaria (e.g., P. falciparum, P. vivax, P. Malariae and/or P. ovale),JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV proteinvaccine, a live attenuated Malaria (e.g., P. falciparum, P. vivax, P.Malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKVand/or YFV vaccine, an inactivated Malaria (e.g., P. falciparum, P.vivax, P. Malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV,DENV, ZIKV and/or YFV vaccine, or a Malaria (e.g., P. falciparum, P.vivax, P. Malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV,DENV, ZIKV and/or YFV VLP vaccine. In some embodiments, the effectiveamount is a dose equivalent to 2-1000-fold reduction in the standard ofcare dose of a recombinant Malaria (e.g., P. falciparum, P. vivax, P.Malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKVand/or YFV protein vaccine, wherein the anti-antigenic polypeptideantibody titer produced in the subject is equivalent to ananti-antigenic polypeptide antibody titer produced in a control subjectadministered the standard of care dose of a recombinant Malaria (e.g.,P. falciparum, P. vivax, P. Malariae and/or P. ovale), JEV, WNV, EEEV,VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV protein vaccine, a purifiedMalaria (e.g., P. falciparum, P. vivax, P. Malariae and/or P. ovale),JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV proteinvaccine, a live attenuated Malaria (e.g., P. falciparum, P. vivax, P.Malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKVand/or YFV vaccine, an inactivated Malaria (e.g., P. falciparum, P.vivax, P. Malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV,DENV, ZIKV and/or YFV vaccine, or a Malaria (e.g., P. falciparum, P.vivax, P. Malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV,DENV, ZIKV and/or YFV VLP vaccine.

In some embodiments, the control is an anti-antigenic polypeptideantibody titer produced in a subject who has been administered avirus-like particle (VLP) vaccine comprising structural proteins ofMalaria (e.g., P. falciparum, P. vivax, P. Malariae and/or P. ovale),JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV.

In some embodiments, the RNA (e.g., mRNA) vaccine is formulated in aneffective amount to produce an antigen specific immune response in asubject.

In some embodiments, the effective amount is a total dose of 25 μg to1000 μg, or 50 μg to 1000 μg. In some embodiments, the effective amountis a total dose of 100 μg. In some embodiments, the effective amount isa dose of 25 μg administered to the subject a total of two times. Insome embodiments, the effective amount is a dose of 100 μg administeredto the subject a total of two times. In some embodiments, the effectiveamount is a dose of 400 μg administered to the subject a total of twotimes. In some embodiments, the effective amount is a dose of 500 μgadministered to the subject a total of two times.

In some embodiments, the efficacy (or effectiveness) of a RNA (e.g.,mRNA) vaccine is greater than 60%. In some embodiments, the RNA (e.g.,mRNA) polynucleotide of the vaccine is at least one of Malaria (e.g., P.falciparum, P. vivax, P. malariae and/or P. ovale), JEV, WNV, EEEV,VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV antigenic polypeptide.

Vaccine efficacy may be assessed using standard analyses (see, e.g.,Weinberg et al., J Infect Dis. 2010 Jun. 1; 201(11):1607-10). Forexample, vaccine efficacy may be measured by double-blind, randomized,clinical controlled trials. Vaccine efficacy may be expressed as aproportionate reduction in disease attack rate (AR) between theunvaccinated (ARU) and vaccinated (ARV) study cohorts and can becalculated from the relative risk (RR) of disease among the vaccinatedgroup with use of the following formulas:

Efficacy=(ARU−ARV)/ARU×100; and

Efficacy=(1−RR)×100.

Likewise, vaccine effectiveness may be assessed using standard analyses(see, e.g., Weinberg et al., J Infect Dis. 2010 Jun. 1;201(11):1607-10). Vaccine effectiveness is an assessment of how avaccine (which may have already proven to have high vaccine efficacy)reduces disease in a population. This measure can assess the net balanceof benefits and adverse effects of a vaccination program, not just thevaccine itself, under natural field conditions rather than in acontrolled clinical trial. Vaccine effectiveness is proportional tovaccine efficacy (potency) but is also affected by how well targetgroups in the population are immunized, as well as by othernon-vaccine-related factors that influence the ‘real-world’ outcomes ofhospitalizations, ambulatory visits, or costs. For example, aretrospective case control analysis may be used, in which the rates ofvaccination among a set of infected cases and appropriate controls arecompared. Vaccine effectiveness may be expressed as a rate difference,with use of the odds ratio (OR) for developing infection despitevaccination:

Effectiveness=(1−OR)×100.

In some embodiments, the efficacy (or effectiveness) of a RNA (e.g.,mRNA) vaccine is at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, or at least 90%.

In some embodiments, the vaccine immunizes the subject against Malaria(e.g., P. falciparum, P. vivax, P. malariae and/or P. ovale), JEV, WNV,EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV for up to 2 years. Insome embodiments, the vaccine immunizes the subject against Malaria(e.g., P. falciparum, P. vivax, P. malariae and/or P. ovale), JEV, WNV,EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV for more than 2 years,more than 3 years, more than 4 years, or for 5-10 years.

In some embodiments, the subject is about 5 years old or younger. Forexample, the subject may be between the ages of about 1 year and about 5years (e.g., about 1, 2, 3, 4 or 5 years), or between the ages of about6 months and about 1 year (e.g., about 6, 7, 8, 9, 10, 11 or 12 months).In some embodiments, the subject is about 12 months or younger (e.g.,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 months or 1 month). In someembodiments, the subject is about 6 months or younger.

In some embodiments, the subject was born full term (e.g., about 37-42weeks). In some embodiments, the subject was born prematurely, forexample, at about 36 weeks of gestation or earlier (e.g., about 36, 35,34, 33, 32, 31, 30, 29, 28, 27, 26 or 25 weeks). For example, thesubject may have been born at about 32 weeks of gestation or earlier. Insome embodiments, the subject was born prematurely between about 32weeks and about 36 weeks of gestation. In such subjects, a RNA (e.g.,mRNA) vaccine may be administered later in life, for example, at the ageof about 6 months to about 5 years, or older.

In some embodiments, the subject is an adult between the ages of about20 years and about 50 years (e.g., about 20, 25, 30, 35, 40, 45 or 50years old).

In some embodiments, the subject is an elderly subject about 60 yearsold, about 70 years old, or older (e.g., about 60, 65, 70, 75, 80, 85 or90 years old).

In some embodiments, the subject has been exposed to Malaria (e.g., P.falciparum, P. vivax, P. Malariae and/or P. ovale), JEV, WNV, EEEV,VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV (e.g., C. trachomatis); thesubject is infected with Malaria (e.g., P. falciparum, P. vivax, P.Malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKVand/or YFV (e.g., C. trachomatis); or subject is at risk of infection byMalaria (e.g., P. falciparum, P. vivax, P. Malariae and/or P. ovale),JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV (e.g., C.trachomatis).

In some embodiments, the subject is immunocompromised (has an impairedimmune system, e.g., has an immune disorder or autoimmune disorder).

In some embodiments the nucleic acid vaccines described herein arechemically modified. In other embodiments the nucleic acid vaccines areunmodified.

Yet other aspects provide compositions for and methods of vaccinating asubject comprising administering to the subject a nucleic acid vaccinecomprising one or more RNA polynucleotides having an open reading frameencoding a first virus antigenic polypeptide, wherein the RNApolynucleotide does not include a stabilization element, and wherein anadjuvant is not coformulated or co-administered with the vaccine.

In other aspects the invention is a composition for or method ofvaccinating a subject comprising administering to the subject a nucleicacid vaccine comprising one or more RNA polynucleotides having an openreading frame encoding a first antigenic polypeptide wherein a dosage ofbetween 10 μg/kg and 400 μg/kg of the nucleic acid vaccine isadministered to the subject. In some embodiments the dosage of the RNApolynucleotide is 1-5 μg, 5-10μg, 10-15 μg, 15-20 μg, 10-25 μg, 20-25μg, 20-50 μg, 30-50 μg, 40-50 μg, 40-60 μg, 60-80 μg, 60-100 μg, 50-100μg, 80-120 μg, 40-120 μg, 40-150 μg, 50-150 μg, 50-200 μg, 80-200 μg,100-200 μg, 120-250 μg, 150-250 μg, 180-280 μg, 200-300 μg, 50-300 μg,80-300 μg, 100-300 μg, 40-300 μg, 50-350 μg, 100-350 μg, 200-350 μg,300-350 μg, 320-400 μg, 40-380 μg, 40-100 μg, 100-400 μg, 200-400 μg, or300-400 μg per dose. In some embodiments, the nucleic acid vaccine isadministered to the subject by intradermal or intramuscular injection.In some embodiments, the nucleic acid vaccine is administered to thesubject on day zero. In some embodiments, a second dose of the nucleicacid vaccine is administered to the subject on day twenty one.

In some embodiments, a dosage of 25 micrograms of the RNA polynucleotideis included in the nucleic acid vaccine administered to the subject. Insome embodiments, a dosage of 100 micrograms of the RNA polynucleotideis included in the nucleic acid vaccine administered to the subject. Insome embodiments, a dosage of 50 micrograms of the RNA polynucleotide isincluded in the nucleic acid vaccine administered to the subject. Insome embodiments, a dosage of 75 micrograms of the RNA polynucleotide isincluded in the nucleic acid vaccine administered to the subject. Insome embodiments, a dosage of 150 micrograms of the RNA polynucleotideis included in the nucleic acid vaccine administered to the subject. Insome embodiments, a dosage of 400 micrograms of the RNA polynucleotideis included in the nucleic acid vaccine administered to the subject. Insome embodiments, a dosage of 200 micrograms of the RNA polynucleotideis included in the nucleic acid vaccine administered to the subject. Insome embodiments, the RNA polynucleotide accumulates at a 100 foldhigher level in the local lymph node in comparison with the distal lymphnode. In other embodiments the nucleic acid vaccine is chemicallymodified and in other embodiments the nucleic acid vaccine is notchemically modified.

Aspects of the invention provide a nucleic acid vaccine comprising oneor more RNA polynucleotides having an open reading frame encoding afirst antigenic polypeptide, wherein the RNA polynucleotide does notinclude a stabilization element, and a pharmaceutically acceptablecarrier or excipient, wherein an adjuvant is not included in thevaccine. In some embodiments, the stabilization element is a histonestem-loop. In some embodiments, the stabilization element is a nucleicacid sequence having increased GC content relative to wild typesequence.

Aspects of the invention provide nucleic acid vaccines comprising one ormore RNA polynucleotides having an open reading frame encoding a firstantigenic polypeptide, wherein the RNA polynucleotide is present in theformulation for in vivo administration to a host, which confers anantibody titer superior to the criterion for seroprotection for thefirst antigen for an acceptable percentage of human subjects. In someembodiments, the antibody titer produced by the mRNA vaccines of theinvention is a neutralizing antibody titer. In some embodiments theneutralizing antibody titer is greater than a protein vaccine. In otherembodiments the neutralizing antibody titer produced by the mRNAvaccines of the invention is greater than an adjuvanted protein vaccine.In yet other embodiments the neutralizing antibody titer produced by themRNA vaccines of the invention is 1,000-10,000, 1,200-10,000,1,400-10,000, 1,500-10,000, 1,000-5,000, 1,000-4,000, 1,800-10,000,2000-10,000, 2,000-5,000, 2,000-3,000, 2,000-4,000, 3,000-5,000,3,000-4,000, or 2,000-2,500. A neutralization titer is typicallyexpressed as the highest serum dilution required to achieve a 50%reduction in the number of plaques.

Also provided are nucleic acid vaccines comprising one or more RNApolynucleotides having an open reading frame encoding a first antigenicpolypeptide, wherein the RNA polynucleotide is present in a formulationfor in vivo administration to a host for eliciting a longer lasting highantibody titer than an antibody titer elicited by an mRNA vaccine havinga stabilizing element or formulated with an adjuvant and encoding thefirst antigenic polypeptide. In some embodiments, the RNA polynucleotideis formulated to produce neutralizing antibodies within one week of asingle administration. In some embodiments, the adjuvant is selectedfrom a cationic peptide and an immunostimulatory nucleic acid. In someembodiments, the cationic peptide is protamine.

Aspects provide nucleic acid vaccines comprising one or more RNApolynucleotides having an open reading frame comprising at least onechemical modification or optionally no modified nucleotides, the openreading frame encoding a first antigenic polypeptide, wherein the RNApolynucleotide is present in the formulation for in vivo administrationto a host such that the level of antigen expression in the hostsignificantly exceeds a level of antigen expression produced by an mRNAvaccine having a stabilizing element or formulated with an adjuvant andencoding the first antigenic polypeptide.

Other aspects provide nucleic acid vaccines comprising one or more RNApolynucleotides having an open reading frame comprising at least onechemical modification or optionally no modified nucleotides, the openreading frame encoding a first antigenic polypeptide, wherein thevaccine has at least 10 fold less RNA polynucleotide than is requiredfor an unmodified mRNA vaccine to produce an equivalent antibody titer.In some embodiments, the RNA polynucleotide is present in a dosage of25-100 micrograms.

Aspects of the invention also provide a unit of use vaccine, comprisingbetween 10 ug and 400 ug of one or more RNA polynucleotides having anopen reading frame comprising at least one chemical modification oroptionally no modified nucleotides, the open reading frame encoding afirst antigenic polypeptide, and a pharmaceutically acceptable carrieror excipient, formulated for delivery to a human subject. In someembodiments, the vaccine further comprises a cationic lipidnanoparticle.

Aspects of the invention provide methods of creating, maintaining orrestoring antigenic memory to a virus strain in an individual orpopulation of individuals comprising administering to said individual orpopulation an antigenic memory booster nucleic acid vaccine comprising(a) at least one RNA polynucleotide, said polynucleotide comprising atleast one chemical modification or optionally no modified nucleotidesand two or more codon-optimized open reading frames, said open readingframes encoding a set of reference antigenic polypeptides, and (b)optionally a pharmaceutically acceptable carrier or excipient. In someembodiments, the vaccine is administered to the individual via a routeselected from the group consisting of intramuscular administration,intradermal administration and subcutaneous administration. In someembodiments, the administering step comprises contacting a muscle tissueof the subject with a device suitable for injection of the composition.In some embodiments, the administering step comprises contacting amuscle tissue of the subject with a device suitable for injection of thecomposition in combination with electroporation.

Aspects of the invention provide methods of vaccinating a subjectcomprising administering to the subject a single dosage of between 25ug/kg and 400 ug/kg of a nucleic acid vaccine comprising one or more RNApolynucleotides having an open reading frame encoding a first antigenicpolypeptide in an effective amount to vaccinate the subject.

Other aspects provide nucleic acid vaccines comprising one or more RNApolynucleotides having an open reading frame comprising at least onechemical modification, the open reading frame encoding a first antigenicpolypeptide, wherein the vaccine has at least 10 fold less RNApolynucleotide than is required for an unmodified mRNA vaccine toproduce an equivalent antibody titer. In some embodiments, the RNApolynucleotide is present in a dosage of 25-100 micrograms.

Other aspects provide nucleic acid vaccines comprising an LNP formulatedRNA polynucleotide having an open reading frame comprising no nucleotidemodifications (unmodified), the open reading frame encoding a firstantigenic polypeptide, wherein the vaccine has at least 10 fold less RNApolynucleotide than is required for an unmodified mRNA vaccine notformulated in a LNP to produce an equivalent antibody titer. In someembodiments, the RNA polynucleotide is present in a dosage of 25-100micrograms.

The data presented in the Examples demonstrate significant enhancedimmune responses using the formulations of the invention. Bothchemically modified and unmodified RNA vaccines are useful according tothe invention. Surprisingly, in contrast to prior art reports that itwas preferable to use chemically unmodified mRNA formulated in a carrierfor the production of vaccines, it is described herein that chemicallymodified mRNA-LNP vaccines required a much lower effective mRNA dosethan unmodified mRNA, i.e., tenfold less than unmodified mRNA whenformulated in carriers other than LNP. Both the chemically modified andunmodified RNA vaccines of the invention produce better immune responsesthan mRNA vaccines formulated in a different lipid carrier.

In other aspects the invention encompasses a method of treating anelderly subject age 60 years or older comprising administering to thesubject a nucleic acid vaccine comprising one or more RNApolynucleotides having an open reading frame encoding a virus antigenicpolypeptide in an effective amount to vaccinate the subject.

In other aspects the invention encompasses a method of treating a youngsubject age 17 years or younger comprising administering to the subjecta nucleic acid vaccine comprising one or more RNA polynucleotides havingan open reading frame encoding a virus antigenic polypeptide in aneffective amount to vaccinate the subject.

In other aspects the invention encompasses a method of treating an adultsubject comprising administering to the subject a nucleic acid vaccinecomprising one or more RNA polynucleotides having an open reading frameencoding a virus antigenic polypeptide in an effective amount tovaccinate the subject.

In some aspects the invention is a method of vaccinating a subject witha combination vaccine including at least two nucleic acid sequencesencoding antigens wherein the dosage for the vaccine is a combinedtherapeutic dosage wherein the dosage of each individual nucleic acidencoding an antigen is a sub therapeutic dosage. In some embodiments,the combined dosage is 25 micrograms of the RNA polynucleotide in thenucleic acid vaccine administered to the subject. In some embodiments,the combined dosage is 100 micrograms of the RNA polynucleotide in thenucleic acid vaccine administered to the subject. In some embodimentsthe combined dosage is 50 micrograms of the RNA polynucleotide in thenucleic acid vaccine administered to the subject. In some embodiments,the combined dosage is 75 micrograms of the RNA polynucleotide in thenucleic acid vaccine administered to the subject. In some embodiments,the combined dosage is 150 micrograms of the RNA polynucleotide in thenucleic acid vaccine administered to the subject. In some embodiments,the combined dosage is 400 micrograms of the RNA polynucleotide in thenucleic acid vaccine administered to the subject. In some embodiments,the sub therapeutic dosage of each individual nucleic acid encoding anantigen is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20 micrograms. In other embodiments the nucleic acid vaccineis chemically modified and in other embodiments the nucleic acid vaccineis not chemically modified.

The RNA polynucleotide is one of SEQ ID NO: 1-12, 18-21, 30-39, 48-51,55-58, 56, 65-96, 118-155, 223-256 or 376-401 and includes at least onechemical modification. In other embodiments the RNA polynucleotide isone of SEQ ID NO: 1-12, 18-21, 30-39, 48-51, 55-58, 56, 65-96, 118-155,223-256 or 376-401 and does not include any nucleotide modifications, oris unmodified. In yet other embodiments the at least one RNApolynucleotide encodes an antigenic protein of any of SEQ ID NO: 13-17,22-29, 44-47, 52-54, 59-64, 97-117, 156-222, 469, 259-291 or 402-413 andincludes at least one chemical modification. In other embodiments theRNA polynucleotide encodes an antigenic protein of any of SEQ ID NO:13-17, 22-29, 44-47, 52-54, 59-64, 97-117, 156-222, 469, 259-291 or402-413 and does not include any nucleotide modifications, or isunmodified.

In preferred aspects, vaccines of the invention (e.g., LNP-encapsulatedmRNA vaccines) produce prophylactically- and/ortherapeutically—efficacious levels, concentrations and/or titers ofantigen-specific antibodies in the blood or serum of a vaccinatedsubject. As defined herein, the term antibody titer refers to the amountof antigen-specific antibody produces in s subject, e.g., a humansubject. In exemplary embodiments, antibody titer is expressed as theinverse of the greatest dilution (in a serial dilution) that still givesa positive result. In exemplary embodiments, antibody titer isdetermined or measured by enzyme-linked immunosorbent assay (ELISA). Inexemplary embodiments, antibody titer is determined or measured byneutralization assay, e.g., by microneutralization assay. In certainaspects, antibody titer measurement is expressed as a ratio, such as1:40, 1:100, etc.

In exemplary embodiments of the invention, an efficacious vaccineproduces an antibody titer of greater than 1:40, greater that 1:100,greater than 1:400, greater than 1:1000, greater than 1:2000, greaterthan 1:3000, greater than 1:4000, greater than 1:500, greater than1:6000, greater than 1:7500, greater than 1:10000. In exemplaryembodiments, the antibody titer is produced or reached by 10 daysfollowing vaccination, by 20 days following vaccination, by 30 daysfollowing vaccination, by 40 days following vaccination, or by 50 ormore days following vaccination. In exemplary embodiments, the titer isproduced or reached following a single dose of vaccine administered tothe subject. In other embodiments, the titer is produced or reachedfollowing multiple doses, e.g., following a first and a second dose(e.g., a booster dose.)

In exemplary aspects of the invention, antigen-specific antibodies aremeasured in units of μg/ml or are measured in units of IU/L(International Units per liter) or mIU/ml (milli International Units perml). In exemplary embodiments of the invention, an efficacious vaccineproduces >0.5 μg/ml, >0.1 μg/ml, >0.2 μg/ml, >0.35 μg/ml, >0.5 μg/ml, >1μg/ml, >2 μg/ml, >5 μg/ml or >10 μg/ml. In exemplary embodiments of theinvention, an efficacious vaccine produces >10 mIU/ml, >20 mIU/ml, >50mIU/ml, >100 mIU/ml, >200 mIU/ml, >500 mIU/ml or >1000 mIU/ml. Inexemplary embodiments, the antibody level or concentration is producedor reached by 10 days following vaccination, by 20 days followingvaccination, by 30 days following vaccination, by 40 days followingvaccination, or by 50 or more days following vaccination. In exemplaryembodiments, the level or concentration is produced or reached followinga single dose of vaccine administered to the subject. In otherembodiments, the level or concentration is produced or reached followingmultiple doses, e.g., following a first and a second dose (e.g., abooster dose.) In exemplary embodiments, antibody level or concentrationis determined or measured by enzyme-linked immunosorbent assay (ELISA).In exemplary embodiments, antibody level or concentration is determinedor measured by neutralization assay, e.g., by microneutralization assay.

The details of various embodiments of the disclosure are set forth inthe description below. Other features, objects, and advantages of thedisclosure will be apparent from the description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows data from an immunogenicity experiment in which mice wereimmunized with JEV prME mRNA vaccine. The data show that immunization ofmice with JEV mRNA vaccine at 10 μg, 2 μg and 0.5 μg doses producesneutralizing antibodies measured between 10² to 10⁴ PRNT50 titers.

FIG. 2 shows a histogram indicating intracellular detection of ZIKA prMEprotein using human serum containing anti-ZIKV antigen antibodies.

FIG. 3 shows the results of detecting prME protein expression inmammalian cells with fluorescence-activated cell sorting (FACS) using aflow cytometer. Cells expressing prME showed higher fluorescenceintensity when stained with anti-ZIKV human serum.

FIG. 4 shows a bar graph of the data provided in FIG. 3.

FIG. 5 shows a reducing SDS-PAGE gel of Zika VLP.

FIG. 6 shows a graph of neutralizing titers obtained from BALB/c miceimmunized with a ZIKV mRNA vaccine encoding prME.

FIGS. 7A-7B show percent animal survival (FIG. 7A) and percent weightchange (FIG. 7B) in animals following administration of two differentdoses of a ZIKV RNA vaccine comprising mRNA encoding ZIKV prME.

FIGS. 8A-8C show Dengue Virus MHC I T cell epitopes. The sequences, fromleft to right correspond to SEQ ID NO: 365-366 (FIG. 8A), 367-368 (FIG.8B), and 369-370 (FIG. 8C).

FIGS. 9A-9C show Dengue Virus MHC II T cell epitopes. The sequences,from left to right, correspond to SEQ ID NO: 371-372 (FIG. 9A), 373-374(FIG. 9B), and 375 (FIG. 9C).

FIG. 10 is a graph depicting the results of an ELISPOT assay ofdengue-specific peptides.

FIG. 11 is a graph depicting the results of an ELISPOT assay ofdengue-specific peptides.

FIG. 12 is a schematic of a bone marrow/liver/thymus (BLT) mouse anddata on human CD8 T cells stimulated with Dengue peptide epitope.

FIGS. 13A and 13B shows the results of an Intracellular CytokineStaining assay performed in PBMC cells.

FIG. 14A shows FACS analyses of cells expressing DENV2 prMEs usingdifferent antibodies against Dengue envelope protein. Numbers in theupper right corner of each plot indicate mean fluorescent intensity.FIG. 14B shows a repeat of staining in triplicate and in two differentcell lines (HeLa and 293T).

FIG. 15 is a graph showing the kinetics of OVA peptide presentation inJawsii cells. All mRNAs tested are formulated in MC3 lipidnanoparticles.

FIG. 16 is a graph showing the Mean Fluorescent Intensity (MFI) ofantibody binding to DENV-1, 2, 3, and 4 prME epitopes presented on thecell surface.

FIGS. 17A-17D are graphs showing the design and the results of achallenge study in AG129 mice. FIG. 17A shows the immunization,challenge, and serum collection schedules. FIG. 17B shows the survivalof the AG129 mice challenged with Dengue D2Y98P virus after beingimmunized with the indicated DENV mRNA vaccines. All immunized micesurvived 11 days post infection, while the unimmunized (control) micedied. FIGS. 17C and 17D show the weight loss of the AG129 mice postinfection. Vaccine 1, 7, 8, or 9 correspond to DENV vaccine construct22, 21, 23, or 24 of the present disclosure, respectively.

FIG. 18 is a graph showing the results of an in vitro neutralizationassay using serum from mice immunized with the DENV mRNA vaccines inFIGS. 17A-17D.

FIGS. 19A-19I are graphs showing the results of a challenge study inAG129 mice. The challenge study design is shown in Table 46. FIGS.19A-19F show the survival, weight loss, and heath score of the AG129mice challenged with D2Y98P virus after being immunized with the DENVmRNA vaccine groups 1-12 in Table 46. FIGS. 19G-19I show the survival,weight loss, and heath score of the AG129 mice challenged with D2Y98Pvirus after being immunized with the DENV mRNA vaccine groups 13-19 inTable 46.

FIG. 20 shows CHIKV envelope protein detection of lysate in HeLa cells16 hours post-transfection.

FIG. 21A is a graph showing the survival rates of AG129 mice vaccinatedwith a single 2 μg dose or two 2 μg doses of Chikungunya E1 antigenadministered either intramuscularly or intradermally. FIG. 21B is agraph showing the percent weight loss of AG129 mice vaccinated with asingle 2 μg dose or two 2 μg doses of Chikungunya E1 antigenadministered either intramuscularly or intradermally. FIG. 21C is agraph showing the health scores of AG129 mice vaccinated with a single 2μg dose or two 2 μg doses of Chikungunya E1 antigen administered eitherintramuscularly or intradermally.

FIG. 22A is a graph showing the survival rates of AG129 mice vaccinatedwith a single 2 μg dose or two 2 μg doses of Chikungunya E2 antigenadministered either intramuscularly or intradermally. FIG. 22B is agraph showing the percent weight loss of AG129 mice vaccinated with asingle 2 μg dose or two 2 μg doses of Chikungunya E2 antigenadministered either intramuscularly or intradermally. FIG. 22C is agraph showing the health scores of AG129 mice vaccinated with a single 2μg dose or two 2 μg doses of Chikungunya E2 antigen administered eitherintramuscularly or intradermally.

FIG. 23A is a graph showing the survival rates of AG129 mice vaccinatedwith a single 2 μg dose or two 2 μg doses of Chikungunya C-E3-E2-6K-E1antigen administered either intramuscularly or intradermally. FIG. 23Bis a graph showing the percent weight loss of AG129 mice vaccinated witha single 2 μg dose or two 2 μg doses of Chikungunya C-E3-E2-6K-E1antigen administered either intramuscularly or intradermally. FIG. 23Cis a graph showing the health scores of AG129 mice vaccinated with asingle 2 μg dose or two 2 μg doses of Chikungunya C-E3-E2-6K-E1 antigenadministered either intramuscularly or intradermally.

FIG. 24A is a graph showing the survival rates of AG129 mice vaccinatedwith a single 10 μg dose or two 10 μg doses of Chikungunya E1 antigenadministered either intramuscularly or intradermally. FIG. 24B is agraph showing the percent weight loss of AG129 mice vaccinated with asingle 10 μg dose or two 10 μg doses of Chikungunya E1 antigenadministered either intramuscularly or intradermally. FIG. 24C is agraph showing the health scores of AG129 mice vaccinated with a single10 μg dose or two 10 μg doses of Chikungunya E1 antigen administeredeither intramuscularly or intradermally.

FIG. 25A is a graph showing the survival rates of AG129 mice vaccinatedwith a single 10 μg dose or two 10 μg doses of Chikungunya E2 antigenadministered either intramuscularly or intradermally. FIG. 25B is agraph showing the percent weight loss of AG129 mice vaccinated with asingle 10 μg dose or two 10 μg doses of Chikungunya E2 antigenadministered either intramuscularly or intradermally. FIG. 25C is agraph showing the health scores of AG129 mice vaccinated with a single10 μg dose or two 10 μg doses of Chikungunya E2 antigen administeredeither intramuscularly or intradermally.

FIG. 26A is a graph showing the survival rates of AG129 mice vaccinatedwith a single 10 μg dose or two 10 μg doses of Chikungunya C-E3-E2-6K-E1antigen administered either intramuscularly or intradermally. FIG. 26Bis a graph showing the percent weight loss of AG129 mice vaccinated witha single 10 μg dose or two 10 μg doses of Chikungunya C-E3-E2-6K-E1antigen administered either intramuscularly or intradermally. FIG. 26Cis a graph showing the health scores of AG129 mice vaccinated with asingle 10 μg dose or two 10 μg doses of Chikungunya C-E3-E2-6K-E1antigen administered either intramuscularly or intradermally.

FIGS. 27A-27B are graphs showing the survival curves from a CHIKVchallenge study in AG129 mice immunized with CHIKV mRNA vaccines in 10μg, 2 μg, or 0.04 μg doses. Mice were divided into 14 groups (1-4 and7-16, n=5). FIG. 27A shows the survival curve of mice groups 1-4 and 7-9challenged on day 56 post immunization. FIG. 27B shows the survivalcurve of mice groups 10-16 challenged on day 112 post immunization.Survival curves were plotted as “percent survival” versus “days postinfection.” See also Table 63 for survival percentage.

FIGS. 28A-28B are graphs showing the weight changes post challenge inAG129 mice immunized with CHIKV mRNA vaccines. FIG. 28A shows the weightchange of mice groups 1-4 and 7-9 challenged on day 56 postimmunization. FIG. 28B shows the weight changes of mice groups 10-16challenged on day 112 post immunization. Initial weights were assessedon individual mice on study Day 0 and daily thereafter. The mean percentweights for each group compared to their percent weight on Day 0(baseline) were plotted against “days post-infection”. Error barsrepresent the standard deviation (SD).

FIGS. 29A-29B are graphs showing the post challenge heath scores ofAG129 mice immunized with CHIKV mRNA vaccines. FIG. 29A shows the healthscores of mice groups 1-4 and 7-9 challenged on day 56 postimmunization. FIG. 29B shows the health score of mice groups 10-16challenged on day 112 post immunization. The mean health scores for eachgroup were plotted against “days post infection” and error barsrepresent the SD. Mean health scores were calculated based onobservations described in Table 51.

FIGS. 30A-30C are graphs showing the antibody titers measured by ELISAassays in the serum of AG129 mice (groups 1-4 and 7-9) 28 days postimmunization with CHIKV mRNA vaccines. FIG. 30A shows the serum antibodytiters against CHIKV E1 protein. FIG. 30B shows the serum antibodytiters against CHIKV E2 protein. FIG. 30C shows the serum antibodytiters against CHIKV lysate.

FIGS. 31A-31C are graphs showing the antibody titers measured by ELISAassays in the serum of AG129 mice (groups 10-16) 28 days postimmunization with CHIKV mRNA vaccine. FIG. 31A shows the serum antibodytiters against CHIKV E1 protein. FIG. 31B shows the serum antibodytiters against CHIKV E2 protein. FIG. 31C shows the serum antibodytiters against CHIKV lysate.

FIGS. 32A-32C are graphs showing the antibody titers measured by ELISAassays in the serum of AG129 mice (groups 10-16) 56 days postimmunization with CHIKV mRNA vaccine. FIG. 32A shows the serum antibodytiters against CHIKV E1 protein. FIG. 32B shows the serum antibodytiters against CHIKV E2 protein. FIG. 32C shows the serum antibodytiters against CHIKV lysate.

FIGS. 33A-33C are graphs showing the antibody titers measured by ELISAassays in the serum of AG129 mice (groups 10-16) 112 days postimmunization with CHIKV mRNA vaccine. FIG. 33A shows the serum antibodytiters against CHIKV E1 protein. FIG. 33B shows the serum antibodytiters against CHIKV E2 protein. FIG. 33C shows the serum antibodytiters against CHIKV lysate.

FIG. 34 shows a set of graphs depicting results of an ELISA assay toidentify the amount of antibodies produced in AG129 mice in response tovaccination with mRNA encoding secreted CHIKV E1 structural protein,secreted CHIKV E2 structural protein, or CHIKV full structuralpolyprotein C-E3-E2-6k-E1 at a dose of 10 μg or 2 μg at 28 days postimmunization.

FIG. 35 shows a set of graphs depicting results of an ELISA assay toidentify the amount of antibodies produced in AG129 mice in response tovaccination with mRNA encoding secreted CHIKV E1 structural protein,secreted CHIKV E2 structural protein, or CHIKV full structuralpolyprotein C-E3-E2-6k-E1 at a dose of 10 μg or 2 μg at 28 days postimmunization. The two panels depict different studies.

FIG. 36 is a graph depicting comparison of ELISA titers from the data ofFIG. 34 to survival in the data of FIG. 35 left panel.

FIG. 37 shows a set of graphs depicting efficacy results in mice inresponse to vaccination with mRNA encoding CHIKV full structuralpolyprotein C-E3-E2-6k-E1 at a dose of 10 μg (left panels), 2 μg (middlepanels) or 0.4 μg (right panels) at 56 days (top panels) or 112 days(bottom panels) post-immunization.

FIG. 38 shows a set of graphs depicting amount of neutralizing antibodyproduced in mice in response to vaccination with mRNA encoding CHIKVfull structural polyprotein C-E3-E2-6k-E1 at a dose of 10 μg, 2 μg, or0.4 μg at 56 days post immunization.

FIG. 39 shows a set of graphs depicting binding antibody produced inmice in response to vaccination with mRNA encoding CHIKV full structuralpolyprotein C-E3-E2-6k-E1 at a dose of 10 μg, 2 μg, or 0.4 μg at 56 dayspost immunization (top panels) and the corresponding correlation betweenbinding and neutralizing antibodies (bottom panels).

FIG. 40 shows a set of graphs depicting amount of neutralizing antibodyproduced in A129 mice in response to vaccination with mRNA encodingCHIKV full structural polyprotein C-E3-E2-6k-E1 at a dose of 10 μg, 2μg, or 0.4 μg at 56 days post immunization against three differentstrains of CHIKV, African—Senegal (left panel), La Reunion (middlepanel) and CDC CAR (right panel).

FIG. 41 shows a graph depicting neutralizing antibodies against CHIKVS27 strain.

FIG. 42 is a graph depicting antibody titer against CHIKV lysate post3rd vaccination 10 with the mRNA vaccine in Sprague Dawley rats.

FIG. 43 shows a set of graphs depicting antibody titers followingvaccination of mice with mRNA encoded CHIKV polyprotein (C-E3-E2-6K-E1).

FIG. 44 shows a set of plots depicting cytokine secretion and T-cellactivation following vaccination of mice with mRNA encoded CHIKVpolyprotein (C-E3-E2-6K-E1).

FIGS. 45A-45B show a set of graphs depicting CD8+ T cell activationfollowing vaccination of mice with mRNA encoded CHIKV polyprotein(C-E3-E2-6K-E1).

FIG. 46 shows a set of graphs depicting binding antibody titers againstCHIKV lysate (upper graph) and neutralizing titers against 37997 CHIKV.The vaccine induces a robust antibody response in non-human primates(NHPs).

FIG. 47 shows a set of graphs depicting a robust CD4 response to a CHIKVvaccine in NHPs.

DETAILED DESCRIPTION

Vaccines containing antigens from more than one pathogenic organismwithin a single dose are referred to as “multivalent” or “combination”vaccines. While various combination vaccines have been approved forhuman use in several countries, including trivalent vaccines forprotecting against diphtheria, tetanus and pertussis (“DTP” vaccines)and trivalent vaccines for protecting against measles, mumps and rubella(“MMR” vaccines), combination vaccines are more complex and areassociated with more problems than monovalent vaccines. For instance,current combination vaccines can include relatively high amounts ofaluminum salts as adjuvants which causes concern to some patientsdespite empirical safety studies. Additionally, the well-documentedphenomenon of antigenic competition (or interference) complicates thedevelopment of multi-component vaccines. Antigenic interference refersto the observation that administering multiple antigens often results ina diminished response to certain antigens relative to the immuneresponse observed when such antigens are administered individually. Thecombination RNA vaccines of the invention can be designed to encode two,three, four, five or more, antigens against multiple pathogenicorganisms, while avoiding a number of the problems associated withtraditional combination vaccines.

Travelers facing a particular geographic viral threat would also benefitfrom vaccination with a combination vaccine of the invention. Thetraveler's vaccine may be tailored based on the prevalence of particularviral diseases in the destination location. For instance a combinationvaccine including WNV, SINV, VEEV, and EEEV would be particularlybeneficial.

Embodiments of the present disclosure provide RNA (e.g., mRNA) vaccinesthat are useful for vaccinating against multiple pathogens. Thecombination vaccines of the present disclosure encode antigens frommultiple pathogens (e.g., bacteria, arboviruses, alphaviruses andflaviviruses), including but not limited to Plasmodium (e.g., P.falciparum, P. vivax, P. Malariae and/or P. ovale), JapaneseEncephalitis Virus (JEV), West Nile Virus (WNV), Eastern EquineEncephalitis (EEEV), Venezuelan Equine Encephalitis Virus (VEEV),Sindbis Virus (SINV), Chikungunya Virus (CHIKV), Dengue Virus (DENV),Zika Virus (ZIKV) and/or Yellow Fever Virus (YFV) antigenic polypeptide.

Thus, the present disclosure provides, in some embodiments, vaccinesthat comprise RNA (e.g., mRNA) polynucleotides encoding a Malaria (e.g.,P. falciparum, P. vivax, P. Malariae and/or P. ovale), JEV, WNV, EEEV,VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV antigenic polypeptide. Thepresent disclosure also provides, in some embodiments, combinationvaccines that comprise at least one RNA (e.g., mRNA) polynucleotideencoding at least two antigenic polypeptides selected from Malaria(e.g., P. falciparum, P. vivax, P. Malariae and/or P. ovale), JEV, WNV,EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and YFV antigenic polypeptides. Alsoprovided herein are methods of administering the RNA (e.g., mRNA)vaccines, methods of producing the RNA (e.g., mRNA) vaccines,compositions (e.g., pharmaceutical compositions) comprising the RNA(e.g., mRNA) vaccines, and nucleic acids (e.g., DNA) encoding the RNA(e.g., mRNA) vaccines. In some embodiments, a RNA (e.g., mRNA) vaccinecomprises an adjuvant, such as a flagellin adjuvant, as provided herein.

The RNA (e.g., mRNA) vaccines (e.g., Malaria (e.g., P. falciparum, P.vivax, P. Malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV,DENV, ZIKV and/or YFV RNA vaccines), in some embodiments, may be used toinduce a balanced immune response, comprising both cellular and humoralimmunity, without many of the risks associated with DNA vaccination.

The entire contents of International Application No. PCT/US2015/02740 isincorporated herein by reference.

Malaria

Malaria is an infectious disease caused by protozoan parasites from thePlasmodium family. Anopheles mosquitoes transmit Malaria, and they musthave been infected through a previous blood meal taken from an infectedperson. When a mosquito bites an infected person, a small amount ofblood is taken in and contains microscopic Malaria parasites. There arefour main types of Malaria which infect humans: Plasmodium falciparum,P. vivax, P. Malariae and P. ovale. Falciparum Malaria is the mostdeadly type. Many Malaria parasites are now immune to the most commondrugs used to treat the disease.

Embodiments of the present disclosure provide RNA (e.g., mRNA) vaccinesthat include polynucleotide encoding a Plasmodium antigen. Malariaparasites are microorganisms that belong to the genus Plasmodium. Thereare more than 100 species of Plasmodium, which can infect many animalspecies such as reptiles, birds, and various mammals. Four species ofPlasmodium have long been recognized to infect humans in nature,including P. falciparum, P. vivax, P. Malariae and P. ovale. Inaddition, there is one species that naturally infects macaques which hasrecently been recognized to be a cause of zoonotic Malaria in humans.

Malaria RNA (e.g., mRNA) vaccines, as provided herein may be used toinduce a balanced immune response, comprising both cellular and humoralimmunity, without many of the risks associated with DNA vaccination.

P. falciparum infects humans and is found worldwide in tropical andsubtropical areas. It is estimated that every year approximately 1million people are killed by P. falciparum, especially in Africa wherethis species predominates. P. falciparum can cause severe Malariabecause it multiples rapidly in the blood, and can thus cause severeblood loss (anemia). In addition, the infected parasites can clog smallblood vessels. When this occurs in the brain, cerebral Malaria results,a complication that can be fatal. Some embodiments of the presentdisclosure provide Malaria vaccines that include at least one RNA (e.g.,mRNA) polynucleotide having an open reading frame encoding at least oneP. falciparum antigenic polypeptide or an immunogenic fragment thereof(e.g., an immunogenic fragment capable of raising an immune response toP. falciparum).

P. vivax infects humans and is found mostly in Asia, Latin America, andin some parts of Africa. Because of the population densities, especiallyin Asia, it is probably the most prevalent human Malaria parasite. P.vivax (as well as P. ovale) has dormant liver stages (“hypnozoites”)that can activate and invade the blood (“relapse”) several months oryears after the infecting mosquito bite. Some embodiments of the presentdisclosure provide Malaria vaccines that include at least RNA (e.g.,mRNA) polynucleotide having an open reading frame encoding at least oneP. vivax antigenic polypeptide or an immunogenic fragment thereof (e.g.,an immunogenic fragment capable of raising an immune response to P.vivax).

P. ovale infects humans and is found mostly in Africa (especially WestAfrica) and the islands of the western Pacific. It is biologically andmorphologically very similar to P. vivax. However, differently from P.vivax, it can infect individuals who are negative for the Duffy bloodgroup, which is the case for many residents of sub-Saharan Africa. Thisexplains the greater prevalence of P. ovale (rather than P. vivax) inmost of Africa. Some embodiments of the present disclosure provideMalaria vaccines that include at least one RNA (e.g., mRNA)polynucleotide having an open reading frame encoding at least one P.ovale antigenic polypeptide or an immunogenic fragment thereof (e.g., animmunogenic fragment capable of raising an immune response to P. ovale).

P. Malariae infects humans and is found worldwide. It is the only humanMalaria parasite species that has a quartan cycle (three-day cycle). Thethree other species that infect human have a tertian, two-day cycle. Ifuntreated, P. Malariae causes a long-lasting, chronic infection that insome cases can last a lifetime. In some chronically infected patients P.Malariae can cause serious complications such as the nephrotic syndrome.Some embodiments of the present disclosure provide Malaria vaccines thatinclude at least one RNA (e.g., mRNA) polynucleotide having an openreading frame encoding at least one P. Malariae antigenic polypeptide oran immunogenic fragment thereof (e.g., an immunogenic fragment capableof raising an immune response to P. Malariae).

P. knowlesi is found throughout Southeast Asia as a natural pathogen oflong-tailed and pig-tailed macaques. It has recently been shown to be asignificant cause of zoonotic Malaria in that region, particularly inMalaysia. P. knowlesi has a 24-hour replication cycle and so can rapidlyprogress from an uncomplicated to a severe infection; fatal cases havebeen reported.

In some embodiments, an antigenic polypeptide is any antigen that isexpressed on the sporozoite or other pre-erythrocytic stage of aPlasmodium parasite, such as the liver stage. For example, an antigenic;polypeptide may be a circumsporozoite (CS) protein, liver stageantigen-1 (LSA1) (see, e.g., WO2004/044167 and Cummings J F et al.Vaccine 2010; 28:5135-44, incorporated herein by reference), liver stageantigen-3 (LSA-3) (see, e.g., EP 0 570 489 and EP 0 833 917,incorporated herein by reference), Pfs 16 kD (see, e.g., WO 91/18922 andEP 597 843), Exported antigen-1 (Exp-1) (described for example inMeraldi et al. Parasite Immunol 2002; 24(3):141, incorporated herein byreference), sporozoite-threonine-asparagine-rich protein (STARP),sporozoite and liver stage antigen (SALSA), thrombospondin relatedanonymous protein (TRAP) (see, e.g., WO 90/01496, WO 91/11516 and WO92/11868, incorporated herein by reference) and apical merozoiteantigen-1 (AMA1) (see, e.g., EP 0 372 019 and Remargue E J et al. Trendsin Parasitology 2007; 24(2):74-84, incorporated herein by reference)which has recently been shown to be present at the liver stage (inaddition to the erythrocytic stage), and merozoite surface protein-1(MSP1) (see, e.g., Reed Z H et al. Vaccine 2009; 27:1651-60,incorporated herein by reference). An antigenic polypeptide may be theentire protein, an immunogenic fragment thereof, or a derivative thereofof any of the foregoing antigens. Immunogenic fragments of Malariaantigens are known, including, for example, the ectodomain from AMA1(see, e.g., WO 02/077195, incorporated herein by reference). Derivativesinclude, for example, fusions with other proteins that may be Malariaproteins or non-Malaria proteins, such as HBsAg. Derivatives of thepresent disclosure are capable of raising an immune response against thenative antigen.

The sporozoite stage of Plasmodium (e.g., P. falciparum or P. vivax) isa potential target of a Malaria vaccine. The major surface protein ofthe sporozoite is circumsporozoite protein (CS protein). The Plasmodium,circumsporozoite protein (CS) is expressed during the sporozoite andearly liver stages of parasitic infection. This protein is involved inthe adhesion of the sporozoite to the hepatocyte and invasion of thehepatocyte. Anti-CS antibodies inhibit parasite invasion and are alsoassociated with a reduced risk of clinical Malaria in some studies.Antibodies raised through immunization with only the conservedAsparagine-Alanine-Asparagine-Proline (NANP) amino acid repeat sequence,the immunodominant B-cell epitope from P. falciparum CS, are capable ofblocking sporozoite invasion of hepatocytes.

CS protein has been cloned, expressed and sequenced for a variety ofstrains, for example for P. falciparum the NF54 strain, clone 3D7(Gaspers et al. Parasitol 1989; 35:185-190, incorporated herein byreference). The protein from strain 3D7 has a central immunodominantrepeat region comprising a tetrapeptide Asn-Ala-Asn-Pro (SEQ ID NO: 434)repeated 40 times and interspersed with four minor repeats of thetetrapeptide Asn-Val-Asp-Pro (SEQ ID NO: 435). In other strains, thenumber of major and minor repeats as well as their relative positionvaries. This central portion is flanked by an N and C terminal portioncomposed of non-repetitive amino acid sequences designated as therepeatless portion of the CS protein.

Some embodiments of the present disclosure provide Malaria vaccines thatinclude at least one RNA (e.g., mRNA) polynucleotide having an openreading frame encoding Plasmodium CS protein or an immunogenic fragmentthereof (e.g., an immunogenic fragment capable of raising an immuneresponse to Plasmodium).

Liver Stage Antigen-1 (LSA1), expressed during Plasmodium falciparumhepatic schizogony is highly conserved, is abundantly expressed fromearly through late schizogony, presumably allowing time for bothcirculating and memory-recalled effector cells to infiltrate the liverand exert their effector function, and it is possible that high titerantibody could act upon the cloud of flocculent liver stage antigenenveloping hepatic merozoites to impede the latter's emergence andsubsequent invasion of erythrocytes. LSA1 is a 230 kDa protein, with alarge central repeat region (over 80 repeats of 17 amino acids each)flanked by two highly conserved N- and C-terminal regions, known tocontain B cell and CD4+ and CD8+ T cell epitopes.

Some embodiments of the present disclosure provide Malaria vaccines thatinclude at least one RNA (e.g., mRNA) polynucleotide having an openreading frame encoding Plasmodium LSA1 or an immunogenic fragmentthereof (e.g., an immunogenic fragment capable of raising an immuneresponse to Plasmodium). In some embodiments, Malaria vaccines includeat least one RNA (e.g., mRNA) polynucleotide having an open readingframe encoding a recombinant protein with full-length C- and N-terminalflanking domains and two of the 17 amino acid repeats from the centralrepeat region, referred to as “LSA-NRC.”

Present on the surface of all known Plasmodium spp., merozoite surfaceprotein 1 (MSP1) is a polypeptide of 190-230 kDa that undergoesprocessing during schizont rupture to produce at least four distinctfragments (83, 28-30, 38-45 and 42 kDa). Further cleavage of thecarboxy-terminal 42-kDa (MSP142) fragment yields a 19-kDa fragment(MSP119), in a process that appears to be critical for merozoiteinvasion. Both MSP₁₄₂ and MSP₁₁₉ regions of P. falciparum areencompassed by the present disclosure.

Thus, in some embodiments, Malaria vaccines include at least one RNA(e.g., mRNA) polynucleotide having an open reading frame encodingPlasmodium MSP1 or an immunogenic fragment thereof (e.g., an immunogenicfragment capable of raising an immune response to Plasmodium).

In some embodiments, Malaria vaccines include at least one RNA (e.g.,mRNA) polynucleotide having an open reading frame encoding PlasmodiumMSP1, MSP3 and AMA1.

Apical membrane antigen 1 (AMA1) is a micronemal protein of apicomplexanparasites that appears to be essential during the invasion of hostcells. Immune responses to Plasmodium AMA1 can have parasite-inhibitoryeffects, both as measured in vitro and in animal challenge models. Firstidentified as an invariant Plasmodium knowlesi merozoite surfaceantigen, AMA1 is believed to be unique to apicomplexan and derives froma single essential gene present in all Plasmodium species.

Some embodiments of the present disclosure provide Malaria vaccines thatinclude at least one RNA (e.g., mRNA) polynucleotide having an openreading frame encoding Plasmodium AMA1 or an immunogenic fragmentthereof (e.g., an immunogenic fragment capable of raising an immuneresponse to Plasmodium).

Japanese Encephalitis Virus (JEV)

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is acommon cause of encephalitis in Asia. Japanese encephalitis (JE) occursthroughout most of Asia and parts of the western Pacific. Among anestimated 35,000-50,000 annual cases, approximately 20%-30% of patientsdie, and 30%-50% of survivors have neurologic or psychiatric sequelae.In endemic countries, JE is primarily a disease of children. However,travel-associated JE, although rare, can occur in a wide portion of thepopulation. JEV is transmitted in an enzootic cycle between mosquitoesand amplifying vertebrate hosts, primarily pigs and wading birds. JEV istransmitted to humans through the bite of an infected mosquito,primarily in rural agricultural areas. In most temperate areas of Asia,JEV transmission is seasonal, and substantial epidemics can occur.

Vaccines available for use against JEV infection include live virusinactivated by such methods as formalin treatment, as well as attenuatedvirus (Tsai et al., in Vaccines (Plotkin, ed.) W.B. Saunders,Philadelphia, Pa., 1994, pp. 671-713). Whole virus vaccines, althougheffective, do have certain problems and/or disadvantages. The virusesare cultivated in mouse brain or in cell culture using mammalian cellsas the host. Such culture methods are cumbersome and expensive.

Embodiments of the present disclosure provide RNA (e.g., mRNA) vaccinesthat include polynucleotide encoding a JEV antigen. JEV is asmall-enveloped virus with a single-stranded, plus-sense RNA genome,consisting of a single open reading frame that codes for a largepolyprotein which is co- and post-translationally cleaved into threestructural (capsid, C; pre-membrane, prM; and envelope, E) and sevennon-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5). TheRNA genome of JEV has a type I cap structure at its 5′-terminus butlacks a poly(A) tail at its 3′ terminus. E protein is involved in anumber of important functions related to virus infection such asreceptor binding and membrane fusion. E protein has been used to raiseantibodies that neutralize virus activity in vitro as well as in vivo.Additionally, sub-viral particles consisting of only the prM and the Eproteins were highly effective in generating protective immune responsein mice against JEV. The ability of various JEV structural andnon-structural proteins to produce an immune response has been examined.(Chen, H. W., et al., 1999. J. Virol. 73:10137-10145.) In view of theseand other studies it has been concluded that the E protein is animportant protein for inducing protective immunity against JEV.

The full-length E protein is membrane anchored. Immunogenic fragments ofthe E protein can be generated by removing the anchor signal. Forinstance, truncated Ea protein wherein a 102-amino acid hydrophobicsequence has been removed from the C-terminus of the protein to generatea 398-amino acid Es protein for immunogenic antigenic fragments. Otherimmunogenic fragments include a secretory form of E protein, as opposedto the anchored protein. Thus immunogenic fragments include thetruncated E protein and the secretory envelope protein (Es) of JEV. JEVantigens may also include one or more non-structural proteins selectedfrom NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5.

Since the envelope (most external portion of a JEV particle) is thefirst to encounter target cells, the present disclosure encompassesantigenic polypeptides associated with the envelope as immunogenicagents. In brief, surface and membrane proteins E, Es, capsid and prM—assingle antigens or in combination with or without adjuvants may be usedas JEV vaccine antigens.

In some embodiments, JEV vaccines comprise RNA (e.g., mRNA) encoding JEVE antigenic polypeptides or immunogenic fragments thereof.

In some embodiments, JEV vaccines comprise RNA (e.g., mRNA) encoding JEVEs antigenic polypeptides or immunogenic fragments thereof.

In some embodiments, JEV vaccines comprise RNA (e.g., mRNA) encoding JEVcapsid antigenic polypeptides or immunogenic fragments thereof.

In some embodiments, JEV vaccines comprise RNA (e.g., mRNA) encoding JEVprM antigenic polypeptides or immunogenic fragments thereof.

In some embodiments, JEV vaccines comprise RNA (e.g., mRNA) encoding JEVNS1 antigenic polypeptides or immunogenic fragments thereof.

In some embodiments, JEV vaccines comprise RNA (e.g., mRNA) encoding JEVantigenic polypeptides having at least 80%, 85%, 90%, 92%, 95%, 96%,97%, 98% or 99% identity with JEV E protein and has receptor bindingand/or membrane fusion activity.

In some embodiments, JEV vaccines comprise RNA (e.g., mRNA) encoding JEVantigenic polypeptides having at least 80%, 85%, 90%, 92%, 95%, 96%,97%, 98% or 99% identity with JEV Es protein and has receptor bindingand/or membrane fusion activity.

In some embodiments, JEV vaccines comprise RNA (e.g., mRNA) encoding JEVantigenic polypeptides having at least 80%, 85%, 90%, 92%, 95%, 96%,97%, 98% or 99% identity with JEV capsid protein having capsid activity.

In some embodiments, JEV vaccines comprise RNA (e.g., mRNA) encoding JEVantigenic polypeptides having at least 80%, 85%, 90%, 92%, 95%, 96%,97%, 98% or 99% identity with JEV prM protein and has activity of animmature virion.

In some embodiments, JEV vaccines comprise RNA (e.g., mRNA) encoding JEVantigenic polypeptides having at least 80%, 85%, 90%, 92%, 95%, 96%,97%, 98% or 99% identity with JEV NS1 protein and has viral replicationand pathogenicity activity.

JEV RNA vaccines, as provided herein may be used to induce a balancedimmune response, comprising both cellular and humoral immunity, withoutmany of the risks associated with DNA vaccination.

West Nile Virus (WNV), Eastern Equine Encephalitis (EEEV), VenezuelanEquine Encephalitis Virus (VEEV), and Sindbis Virus (SINV)

WNV was first isolated in the West Nile region of Uganda, Africa, andbelongs to the Flaviviridae family falvivirus genus. The structure ofvirus particles consists of a spherical structure wherein a capsidprotein (C protein) is bonded to one (+) chain RNA virus gene, and alipid bilayer membrane surrounding the spherical structure. The lipidmembrane includes two kinds of proteins: envelope protein (E protein)and membrane protein (M protein). M protein is produced as a precursorprM protein and cleaved with a protease called furin to become a matureprotein. West Nile virus (WNV) is an important mosquito transmittedvirus which is now native to the U.S.

West Nile fever is a systemic acute fever disease caused by infectionwith WNV. Occasionally, the virus invades and grows in the centralnervous system to cause lethal brain meningitis. WNV is widelydistributed in Africa, Middle East, part of Europe, Russia, India, andIndonesia. The virus is maintained and propagated by an infection ring.The West Nile fever virus is transmitted to birds and mammals by thebites of certain mosquitoes (e.g., Culex, Aedes, Anopheles). Directtransmission may happen from WNV infected subject to healthy subject byoral transmission (prey and transmission through colostrum) andblood/organ vectored transmission. Humans, horses and domestic animalsare hosts. Recently, WNV invaded and was indigenized in the US and hasexpanded since then. A prevalent US strain is West Nile virusNY99-flamingo382-99 strain (Lanciotti, R. S. et al., Science, 286:2333-2337, 1999) (GenBank Accession No. AF196835).

The WNV antigens in the combination RNA vaccine may be derived from aparticular WNV strain, such as NY99 or KEN-3829 or any other strain.Additional WNV strains are known in the art. West Nile virus antigensinclude the following proteins and polyproteins: C (capsid), E(envelope), M (membrane), prM (Pre-membrane), NS2A, NS2B, NS3 prM-E,M-E, prM-M, prM-M-E, and NS2A-NS2B-NS3.

Eastern equine encephalitis virus (EEEV), Western equine encephalitisvirus (WEEV), and Venezuelan equine encephalitis virus (VEEV) aremembers of the Alphavirus genus of the family Togaviridae. The genus iscomprised of at least 27 different arthropod-borne RNA viruses that arefound throughout much of the world. The viruses normally circulate amongavian or rodent hosts through the feeding activities of a variety ofmosquitoes.

EEEV causes encephalitis in humans and equines in epidemic proportions.However, EEEV causes the most severe of the arboviral encephalitides inhumans, with high mortality and severe neurological sequelae insurvivors (Fields Virology, 4.sup.th Ed., Chapter 30 Alphaviruses,[2002] 917-962). The virus is known to be focally endemic along much ofthe Atlantic and Gulf Coasts of North America. It has also been found insouthern Canada, the Caribbean, Central America, the eastern part ofMexico and in large sections of South America. Inland foci exist in theGreat Lakes region and South Dakota in the U.S. as well as the AmazonBasin.

The current EEEV vaccine for veterinary applications in the U.S. is aformalin-inactivated whole virus preparation derived from the PE-6strain (Bartelloni, et al. [1970] Am J. Trop Med Hyg. 19:123-126; Marie,et al. [1970] Am J Trop Med Hyg. 19:119-122). Currently there is nohuman vaccine. The inactivated veterinary vaccine is poorly immunogenic,requires multiple inoculations with frequent boosters and generallyresults in immunity of short duration.

EEEV, SINV, JEV, and CHIKV all have single-stranded, positive sense RNAgenomes. A portion of the genome encodes the viral structural proteinsCapsid, E3, E2, 6K, and E1, each of which are derived by proteolyticcleavage of the product of a single open reading frame. The nucleocapsid(C) protein possesses autoproteotytic activity which cleaves the Cprotein from the precursor protein soon after the ribosome transits thejunction between the C and E3 protein coding sequence. Subsequently, theenvelope glycoproteins E2 and E1 are derived by proteolytic cleavage inassociation with intracellular membranes and form heterodimers. E2initially appears in the infected cell as the precursor protein PE2,which consists of E3 and E2. After extensive glycosylation and transitthrough the endoplasmic reticulum and the Golgi apparatus, E3 is cleavedfrom E2 by the furin protease. Subsequently, the E2/E1 complex istransported to the cell surface where it is incorporated into virusbudding from the plasma membrane. The envelope proteins play animportant role in attachment and fusion to cells.

Sindbis Virus (SINV) is also a member of the Togaviridae family, in thealphavirus subfamily and is transmitted by mosquitoes. Sindbis fever ismost common in South and East Africa, Egypt, Israel, Philippines andparts of Australia. The genome encodes four non-structural proteins atthe 5′ end and the capsid and two envelope proteins at the 3′ end. Thenon-structural proteins are involved in genome replication and theproduction of new genomic RNA and a shorter sub-genomic RNA strand. Theviruses assemble at the host cell surfaces and acquire their envelopethrough budding.

Yellow Fever Virus (YFV)

Along with other viruses in the Flaviviridae family, Yellow fever virusis enveloped and icosahedral with a non-segmented, single-stranded,positive sense RNA genome. It is most closely related to the Sepik virusand is one of the two viruses in clade VIII. In 1927, Yellow fever viruswas the first human virus to be isolated. It is found in tropical areasof Africa and South America. YFV is believed to have originated inAfrica and spread to South America through slave trades in the 17^(th)century. Since then, there have been Yellow fever outbreaks in theAmericas, Africa and Europe. It is transmitted by mosquitoes and hasbeen isolated from a number of species in the genus Aedes (e.g., Aedesaegypti, Aedes africanus or Aedes albopictus). Mosquitos of the genusHaemagogus and Sabethes can also serve as vectors. Studies show that theextrinsic incubation period in mosquitoes is about 10 days. Vertebratehosts of the virus include monkeys and humans.

Forty-seven African and South American countries are either endemic for,or have regions that are endemic from, Yellow fever. It is estimatedthat in 2013 alone, there were 84,000 to 170,000 severe cases of yellowfever and 29,000 to 60,000 deaths associated with Yellow fever.

What is important is not only the number of cases but also the clinicalmanifestation of the cases. After YFV incubates in the body for about 6days, symptoms including fever, muscle pain, backache, headache, loss ofappetite, and nausea or vomiting are observed. In most cases, thesesymptoms disappear after about 4 days. In a small percentage ofpatients, a more toxic phase of the disease is observed within 24 hoursof recovering from the initial symptoms. In this toxic phase, patientsdevelop high fever, jaundice, dark urine and abdominal pain withvomiting. Half of the patients that enter the toxic phase die within 10days.

In some embodiments, YFV vaccines comprise RNA (e.g., mRNA) encoding aYFV antigenic polypeptide having at least 95%, at least 96%, at least97%, at least 98% or at least 99% identity with YFV polyprotein andhaving YFV polyprotein activity, respectively. The YFV polyprotein iscleaved into capsid, precursor membrane, envelope, and non-structuralproteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5).

A protein is considered to have YFV polyprotein activity if, forexample, it facilitates the attachment of the viral envelope to hostreceptors, mediates internalization into the host cell, and aids infusion of the virus membrane with the host's endosomal membrane.

Zika Virus (ZIKV)

Along with other viruses in the Flaviviridae family, Zika virus isenveloped and icosahedral with a non-segmented, single-stranded,positive sense RNA genome. It is most closely related to the Spondwenivirus and is one of the two viruses in the Spondweni virus clade. Thevirus was first isolated in 1947 from a rhesus monkey in the Zika Forestof Uganda, Africa and was isolated for the first time from humans in1968 in Nigeria. From 1951 through 1981, evidence of human infection wasreported from other African countries such as Uganda, Tanzania, Egypt,Central African Republic, Sierra Leone and Gabon, as well as in parts ofAsia including India, Malaysia, the Philippines, Thailand, Vietnam andIndonesia. It is transmitted by mosquitoes and has been isolated from anumber of species in the genus Aedes—Aedes aegypti, Aedes africanus,Aedes apicoargenteus, Aedes furcifer, Aedes luteocephalus and Aedesvitattus. Studies show that the extrinsic incubation period inmosquitoes is about 10 days. The vertebrate hosts of the virus includemonkeys and humans.

As of early 2016, the most widespread outbreak of Zika fever, caused bythe Zika virus, is ongoing primarily in the Americas. The outbreak beganin April 2015 in Brazil, and subsequently spread to other countries inSouth America, Central America, and the Caribbean.

The Zika virus was first linked with newborn microcephaly during theBrazil Zika virus outbreak. In 2015, there were 2,782 cases ofmicrocephaly compared with 147 in 2014 and 167 in 2013. The BrazilianHealth Ministry has reported 4783 cases of suspected microcephaly as ofJan. 30, 2016, an increase of more than 1000 cases from a week earlier.Confirmation of many of the recent cases is pending, and it is difficultto estimate how many cases went unreported before the recent awarenessof the risk of virus infections.

What is important is not only the number of cases but also the clinicalmanifestation of the cases. Brazil is seeing severe cases ofmicrocephaly, which are more likely to be paired with greaterdevelopmental delays. Most of what is being reported out of Brazil ismicrocephaly with other associated abnormalities. The potentialconsequence of this is the fact that there are likely to be subclinicalcases where the neurological sequelae will only become evident as thechildren grow.

Zika virus has also been associated with an increase in a rare conditionknown as Guillain-Barré, where the infected individual becomesessentially paralyzed. During the Zika virus outbreak in FrenchPolynesia, of the 74 patients which had had Zika symptoms, 42 werediagnosed with Guillain-Barré syndrome. In Brazil, 121 cases ofneurological manifestations and Guillain-Barré syndrome (GBS) werereported, all cases with a history of Zika-like symptoms.

The design of preferred Zika vaccine mRNA constructs of the inventionencode prME proteins from the Zika virus intended to produce significantimmunogenicity. The open reading frame comprises a signal peptide (tooptimize expression into the endoplasmic reticulum) followed by the ZikaprME polyprotein sequence. The particular prME sequence used is from aMicronesian strain (2007) that most closely represents a consensus ofcontemporary strain prMEs. This construct has 99% prME sequence identityto the current Brazilian isolates.

Within the Zika family, there is a high level of homology within theprME sequence (>90%) across all strains so far isolated. The high degreeof homology is also preserved when comparing the original isolates from1947 to the more contemporary strains circulating in Brazil in 2015,suggesting that there is “drift” occurring from the original isolates.Furthermore, attenuated virus preparations have providedcross-immunization to all other strains tested, including LatinAmerican/Asian, and African. Overall, this data suggests thatcross-protection of all Zika strains is possible with a vaccine based onprME. In fact, the prM/M and E proteins of ZIKV have a very high level(99%) of sequence conservation between the currently circulating Asiaticand Brazilian viral strains.

The M and E proteins are on the surface of the viral particle.Neutralizing antibodies predominantly bind to the E protein, the preM/Mprotein functions as a chaperone for proper folding of E protein andprevent premature fusion of E protein within acidic compartments alongthe cellular secretory pathway.

Described herein are examples of ZIKV vaccine designs comprising mRNAencoding the both prM/M and E proteins or E protein alone. In someembodiments, the mRNA encodes an artificial signal peptide fused to prMprotein fused to E protein. In some embodiments, the mRNA encodes anartificial signal peptide fused to E protein.

ZIKV vaccine constructs can encode the prME or E proteins from differentstrains, for example, Brazil_isolate_ZikaSPH2015 or ACD75819_Micronesia,having a signal peptide fused to the N-termini of the antigenicprotein(s). In some embodiments, ZIKV vaccines comprise mRNAs encodingantigenic polypeptides having amino acid sequences of SEQ ID NO: 156-222or 469.

Dengue Virus (DENV)

There is no specific treatment for DENV infection, and control of DENVby vaccination has proved elusive, in part, because the pathogenesis ofDHF/DSS is not completely understood. While infection with one serotypeconfers lifelong homotypic immunity, it confers only short term(approximately three to six months) cross protection against heterotypicserotypes. Also, there is evidence that prior infection with one typecan produce an antibody response that can intensify, or enhance, thecourse of disease during a subsequent infection with a differentserotype. The possibility that vaccine components could elicit enhancingantibody responses, as opposed to protective responses, has been a majorconcern in designing and testing vaccines to protect against dengueinfections.

In late 2015 and early 2016, the first dengue vaccine, Dengvaxia(CYD-TDV) by Sanofi Pasteur, was registered in several countries for usein individuals 9-45 years of age living in endemic areas. Issues withthe vaccine include (1) weak protection against DENV1 and DENV2 (<60%efficacy); (2) relative risk of dengue hospitalization among children <9years old (7.5×higher than placebo); (3) immunogenicity not sustainedafter 1-2 years (implying the need for a 4^(th) dose booster); and (4)lowest efficacy against DENV2, which often causes more severeconditions. This latter point is a major weakness with the Dengvaxiavaccine, signaling the need of a new, more effective vaccine effectiveagainst DENV2. Other tetravalent live-attenuated vaccines are underdevelopment in phase II and phase III clinical trials, and other vaccinecandidates (based on subunit, DNA and purified inactivated virusplatforms) are at earlier stages of clinical development, although theability of these vaccine candidates to provide broad serotype protectionhas not been demonstrated.

Embodiments of the present disclosure provide RNA (e.g., mRNA) vaccinesthat include at least one RNA polynucleotide encoding a Dengue virus(DENV) antigen. Dengue virus is a mosquito-borne (Aedes aegypti/Aedesalbopictus) member of the family Flaviviridae (positive-sense,single-stranded RNA virus). The dengue virus genome encodes ten genesand is translated as a single polypeptide which is cut into tenproteins: the capsid, envelope, membrane, and nonstructural proteins(NS1, NS2A, NS2B, NS3, SN4A, NS4B, and NS5 proteins). The virus' mainantigen is DENV envelope (E) protein, which is a component of the viralsurface and is thought to facilitate the binding of the virus tocellular receptors (Heinz et al., Virology. 1983, 126:525). There arefour similar but distinct serotypes of dengue virus (DENV-1, DENV-2,DENV-3, DENV-4, and DENV-5), which result annually in an estimated50-100 million cases of dengue fever and 500,000 cases of the moresevere dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) (Gubleret al., Adv Virus Res. 1999, 53:35-70). The four serotypes showimmunological cross-reactivity, but are distinguishable in plaquereduction neutralization tests and by their respective monoclonalantibodies. The dengue virus E protein includes a serotype-specificantigenic determinant and determinants necessary for virusneutralization (Mason et al., J Gen Virol. 1990, 71:2107-2114).

After inoculation, the dendritic cells become infected and travel tolymph nodes. Monocytes and macrophages are also targeted shortlythereafter. Generally, the infected individual will be protected againsthomotypic reinfection for life; however, the individual will only beprotected against other serotypes for a few weeks or months (Sabin, Am JTrop Med Hyg. 1952, 1:30-50). In fact, DHF/DSS is generally found inchildren and adults infected with a dengue virus serotype differing fromtheir respective primary infection. Thus, it is necessary to develop avaccine that provides immunity to all four serotypes.

The DENV E (envelope) protein is found on the viral surface and plays arole in the initial attachment of the viral particle to the host cell.Several molecules which interact with the viral E protein(ICAM3-grabbing non-integrin, CD209, Rab 5, GRP 78, and the mannosereceptor) are thought to be important factors mediating attachment andviral entry.

The DENV prM (membrane) protein is important in the formation andmaturation of the viral particle. The membrane protein consists of sevenantiparallel β-strands stabilized by three disulfide bonds. Theglycoprotein shell of the mature DENV virion consists of 180 copies eachof the E protein and M protein. The immature virion comprises E and prMproteins, which form 90 heterodimer spikes on the exterior of the viralparticle. The immature viral particle buds into the endoplasmicreticulum and eventually travels via the secretory pathway to the Golgiapparatus. As the virion passes through the trans-Golgi Network (TGN),it is exposed to an acidic environment which causes a conformationalchange in the E protein which causes it to disassociate from the prMprotein and form E homodimers. During the maturation phase, the prpeptide is cleaved from the M peptide by the host protease, furin. The Mprotein then acts as a transmembrane protein under the E-protein shellof the mature virion. The pr peptide remains associated with the Eprotein until the viral particle is released into the extracellularenvironment, acting like a cap covering the hydrophobic fusion loop ofthe E protein until the viral particle has exited the cell.

The DENV NS3 is a serine protease, as well as an RNA helicase andRTPase/NTPase. The protease domain consists of six β-strands arrangedinto two β-barrels formed by residues 1-180 of the protein. Thecatalytic triad (His-51, Asp-75 and Ser-135), is found between these twoβ-barrels, and its activity is dependent on the presence of the NS2Bcofactor which wraps around the NS3 protease domain and becomes part ofthe active site. The remaining NS3 residues (180-618), form the threesubdomains of the DENV helicase. A six-stranded parallel β-sheetsurrounded by four α-helices make up subdomains I and II, and subdomainIII is composed of 4 α-helices surrounded by three shorter α-helices andtwo antiparallel β-strands.

Chikungunya Virus (CHIKV)

Presently, CHIKV is a re-emerging human pathogen that has nowestablished itself in Southeast Asia and has more recently spread toEurope. The Chikungunya virus (CHIKV) was introduced into Asia around1958, and sites of endemic transmission within Southeastern Asia,including the Indian Ocean, were observed through 1996. The CHIKVepidemic moved throughout Asia, reaching Europe and Africa in the early2000s, and was imported via travelers to North America and South Americafrom 2005 to 2007. Sporadic outbreaks are still occurring in severalcountries, such as Italy, infecting naive populations. Singapore, forinstance, experienced two successive waves of Chikungunya virusoutbreaks in January and August 2008. Of the two strain lineages ofCHIKV, the African strain remains enzootic by cycling between mosquitoesand monkeys, but the Asian strain is transmitted directly betweenmosquitoes and humans. This cycle of transmission may have allowed thevirus to become more pathogenic as the reservoir host was eliminated.

In humans, CHIKV causes a debilitating disease characterized by fever,headache, nausea, vomiting, fatigue, rash, muscle pain and joint pain.Following the acute phase of the illness, patients develop severechronic symptoms lasting from several weeks to months, includingfatigue, incapacitating joint pain and polyarthritis.

The re-emergence of CHIKV has caused millions of cases throughoutcountries around the Indian Ocean and in Southeast Asia. Specifically,India, Indonesia, Maldives, Myanmar and Thailand have reported over 1.9million cases since 2005. Globally, human CHIKV epidemics from 2004-2011have resulted in 1.4-6.5 million reported cases, including a number ofdeaths. Thus, CHIKV remains a public threat that constitutes a majorpublic health problem with severe social and economic impact.

Despite significant morbidity and some cases of mortality associatedwith CHIKV infection and its growing prevalence and geographicdistribution, there is currently no licensed CHIKV vaccine or antiviralapproved for human use. Several potential CHIKV vaccine candidates havebeen tested in humans and animals with varying success.

Chikungunya virus is a small (about 60-70 nm diameter), spherical,enveloped, positive-strand RNA virus having a capsid with icosahedralsymmetry. The virion consists of an envelope and a nucleocapsid. Thevirion RNA is infectious and serves as both genome and viral messengerRNA. The genome is a linear, ssRNA(+) genome of 11,805 nucleotides whichencodes two polyproteins that are processed by host and viral proteasesinto non-structural proteins (nsP1, nsP2, nsP3, and RdRpnsP4) necessaryfor RNA synthesis (replication and transcription) and structuralproteins (capsid and envelope proteins C, E3, E2, 6K, and E1) whichattach to host receptors and mediate endocytosis of virus into the hostcell. The E1 and E2 glycoproteins form heterodimers that associate as 80trimeric spikes on the viral surface covering the surface evenly. Theenvelope glycoproteins play a role in attachment to cells. The capsidprotein possesses a protease activity that results in its self-cleavagefrom the nascent structural protein. Following its cleavage, the capsidprotein binds to viral RNA and rapidly assembles into icosahedric coreparticles. The resulting nucleocapsid eventually associates with thecytoplasmic domain of E2 at the cell membrane, leading to budding andformation of mature virions.

E2 is an envelope glycoprotein responsible for viral attachment totarget host cell, by binding to the cell receptor. E2 is synthesized asa p62 precursor which is processed at the cell membrane prior to virionbudding, giving rise to an E2-E1 heterodimer. The C-terminus of E2 isinvolved in budding by interacting with capsid proteins.

E1 is an envelope glycoprotein with fusion activity, which is inactiveas long as E1 is bound to E2 in the mature virion. Following virusattachment to target cell and endocytosis, acidification of the endosomeinduces dissociation of the E1/E2 heterodimer and concomitanttrimerization of the E1 subunits. The E1 trimer is fusion active andpromotes the release of the viral nucleocapsid in the cytoplasm afterendosome and viral membrane fusion.

E3 is an accessory protein that functions as a membranetranslocation/transport signal for E1 and E2.

6K is another accessory protein involved in virus glycoproteinprocessing, cell permeabilization, and the budding of viral particlesLike E3, it functions as a membrane transport signal for E1 and E2.

The CHIKV structural proteins have been shown to be antigenic, whichproteins, fragments, and epitopes thereof are encompassed within theinvention. A phylogenetic tree of Chikungunya virus strains derived fromcomplete concatenated open reading frames for the nonstructural andstructural polyproteins shows key envelope glycoprotein E1 amino acidsubstitutions that facilitated (Indian Ocean lineage) or prevented(Asian lineage) adaptation to Aedes albopictus. There are membrane-boundand secreted forms of E1 and E2, as well as the full length polyproteinantigen (C-E3-E2-6K-E1), which retains the protein's nativeconformation. Additionally, the different Chikungunya genotypes, strainsand isolates can also yield different antigens, which are functional inthe constructs of the invention. For example, there are severaldifferent Chikungunya genotypes: Indian Ocean, East/Central/SouthAfrican (ECSA), Asian, West African, and the Brazilian isolates(ECSA/Asian). There are three main Chikungunya genotype. These are ESCA(East-South-Central Africa), Asia, and West Africa. While sometimesnames differ in publications, all belong to these three geographicalstrains.

The entire contents of International Application No. PCT/US2015/02740 isincorporated herein by reference.

Combination Vaccines

Embodiments of the present disclosure also provide combination RNA(e.g., mRNA) vaccines. A “combination RNA (e.g., mRNA) vaccine” of thepresent disclosure refers to a vaccine comprising at least one (e.g., atleast 2, 3, 4, 5, 6, 7, 8, 9 or 10) RNA (e.g., mRNA) polynucleotidehaving an open reading frame encoding a combination of at least oneMalaria (e.g., P. falciparum, P. vivax, P. malariae and/or P. ovale)antigenic polypeptide, at least one JEV antigenic polypeptide, at leastone WNV antigenic polypeptide, at least one EEEV antigenic polypeptide,at least one VEEV antigenic polypeptide, at least one SINV antigenicpolypeptide, at least on CHIKV antigenic polypeptide, at least one DENVantigenic polypeptide, at least one ZIKV antigenic polypeptide, at leastone YFV antigenic polypeptide, or any combination of two, three, four,five, six, seven, eight, nine, ten or more of the foregoing antigenicpolypeptides.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide, a WNV antigenic polypeptide, a EEEV antigenicpolypeptide, a VEEV antigenic polypeptide, a SINV antigenic polypeptide,a CHIKV antigenic polypeptide, a DENV antigenic polypeptide, a ZIKVantigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide and a JEVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide and a WNVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide and a EEEVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide and a VEEVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide and a SINVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide and a CHIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide and a DENVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide anda WNV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide anda EEEV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide anda VEEV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide anda SINV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide anda CHIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide anda DENV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide anda ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide anda YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide anda EEEV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide anda VEEV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide anda SINV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide anda CHIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide anda DENV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide anda ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide anda YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptideand a VEEV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptideand a SINV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptideand a CHIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptideand a DENV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptideand a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptideand a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a VEEV antigenic polypeptideand a SINV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a VEEV antigenic polypeptideand a CHIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a VEEV antigenic polypeptideand a DENV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a VEEV antigenic polypeptideand a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a VEEV antigenic polypeptideand a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a SINV antigenic polypeptideand a CHIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a SINV antigenic polypeptideand a DENV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a SINV antigenic polypeptideand a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a SINV antigenic polypeptideand a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a CHIKV antigenic polypeptideand a DENV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a CHIKV antigenic polypeptideand a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a CHIKV antigenic polypeptideand a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a DENV antigenic polypeptideand a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a DENV antigenic polypeptideand a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a ZIKV antigenic polypeptideand a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide and a WNV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide and a EEEV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide and a VEEV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide and a SINV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide and a CHIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide and a DENV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide and a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a WNVantigenic polypeptide and a EEEV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a WNVantigenic polypeptide and a VEEV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a WNVantigenic polypeptide and a SINV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a WNVantigenic polypeptide and a CHIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a WNVantigenic polypeptide and a DENV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a WNVantigenic polypeptide and a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a WNVantigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a EEEVantigenic polypeptide and a VEEV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a EEEVantigenic polypeptide and a SINV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a EEEVantigenic polypeptide and a CHIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a EEEVantigenic polypeptide and a DENV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a EEEVantigenic polypeptide and a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a EEEVantigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a VEEVantigenic polypeptide and a SINV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a VEEVantigenic polypeptide and a CHIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a VEEVantigenic polypeptide and a DENV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a VEEVantigenic polypeptide and a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a VEEVantigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, SINVantigenic polypeptide and a CHIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, SINVantigenic polypeptide and a DENV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, SINVantigenic polypeptide and a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, SINVantigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a CHIKVantigenic polypeptide and a DENV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a CHIKVantigenic polypeptide and a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a CHIKVantigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a DENVantigenic polypeptide and a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a DENVantigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a ZIKVantigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide and a EEEV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide and a VEEV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide and a SINV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide and a CHIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide and a DENV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide and a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aEEEV antigenic polypeptide and a VEEV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aEEEV antigenic polypeptide and a SINV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aEEEV antigenic polypeptide and a CHIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aEEEV antigenic polypeptide and a DENV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aEEEV antigenic polypeptide and a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aEEEV antigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aVEEV antigenic polypeptide and a SINV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aVEEV antigenic polypeptide and a CHIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aVEEV antigenic polypeptide and a DENV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aVEEV antigenic polypeptide and a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aVEEV antigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aSINV antigenic polypeptide and a CHIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aSINV antigenic polypeptide and DENV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aSINV antigenic polypeptide and a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aSINV antigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide,CHIKV antigenic polypeptide and a DENV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide,CHIKV antigenic polypeptide and a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide,CHIKV antigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aDENV antigenic polypeptide and a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aDENV antigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aZIKV antigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aEEEV antigenic polypeptide and a VEEV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aEEEV antigenic polypeptide and a SINV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aEEEV antigenic polypeptide and a CHIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aEEEV antigenic polypeptide and a DENV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aEEEV antigenic polypeptide and a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aEEEV antigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding WNV antigenic polypeptide, aVEEV antigenic polypeptide and a SINV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding WNV antigenic polypeptide, aVEEV antigenic polypeptide and a CHIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding WNV antigenic polypeptide, aVEEV antigenic polypeptide and a DENV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding WNV antigenic polypeptide, aVEEV antigenic polypeptide and a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding WNV antigenic polypeptide, aVEEV antigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aSINV antigenic polypeptide and a CHIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aSINV antigenic polypeptide and a DENV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aSINV antigenic polypeptide and a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aSINV antigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aSINV antigenic polypeptide and a CHIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aSINV antigenic polypeptide and a DENV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aSINV antigenic polypeptide and a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aSINV antigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aCHIKV antigenic polypeptide and a DENV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aCHIKV antigenic polypeptide and a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aCHIKV antigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aDENV antigenic polypeptide and a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aDENV antigenic polypeptide and YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aZIKV antigenic polypeptide and YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aVEEV antigenic polypeptide and a SINV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aVEEV antigenic polypeptide and a CHIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aVEEV antigenic polypeptide and a DENV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aVEEV antigenic polypeptide and a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aVEEV antigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aSINV antigenic polypeptide and a CHIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aSINV antigenic polypeptide and a DENV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aSINV antigenic polypeptide and a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aSINV antigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aCHIKV antigenic polypeptide and a DENV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aCHIKV antigenic polypeptide and a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aCHIKV antigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aDENV antigenic polypeptide and a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aDENV antigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aZIKV antigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a VEEV antigenic polypeptide, aSINV antigenic polypeptide and a CHIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a VEEV antigenic polypeptide, aSINV antigenic polypeptide and a DENV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a VEEV antigenic polypeptide, aSINV antigenic polypeptide and a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a VEEV antigenic polypeptide, aSINV antigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a VEEV antigenic polypeptide, aCHIKV antigenic polypeptide and a DENV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a VEEV antigenic polypeptide, aCHIKV antigenic polypeptide and a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a VEEV antigenic polypeptide, aCHIKV antigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a VEEV antigenic polypeptide, aDENV antigenic polypeptide and a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a VEEV antigenic polypeptide, aDENV antigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a VEEV antigenic polypeptide, aZIKV antigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a SINV antigenic polypeptide, aCHIKV antigenic polypeptide and a DENV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a SINV antigenic polypeptide, aCHIKV antigenic polypeptide and a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a SINV antigenic polypeptide, aCHIKV antigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a SINV antigenic polypeptide, aDENV antigenic polypeptide and a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a SINV antigenic polypeptide, aDENV antigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a SINV antigenic polypeptide, aZIKV antigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a CHIKV antigenic polypeptide,a DENV antigenic polypeptide and a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a CHIKV antigenic polypeptide,a DENV antigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a CHIKV antigenic polypeptide,a ZIKV antigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a DENV antigenic polypeptide, aZIKV antigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide, a WNV antigenic polypeptide and a EEEV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide, a WNV antigenic polypeptide and a VEEV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide, a WNV antigenic polypeptide and a SINV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide, a WNV antigenic polypeptide and a CHIKV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide, a WNV antigenic polypeptide and a DENV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide, a WNV antigenic polypeptide and a ZIKV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide, a WNV antigenic polypeptide and a YFV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide, a EEEV antigenic polypeptide and a VEEV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide, a EEEV antigenic polypeptide and a SINV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide, a EEEV antigenic polypeptide and a CHIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide, a EEEV antigenic polypeptide and a DENV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide, a EEEV antigenic polypeptide and a ZIKV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide, a EEEV antigenic polypeptide and a YFV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide and a VEEV antigenic polypeptide and a SINVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide and a VEEV antigenic polypeptide and a CHIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide and a VEEV antigenic polypeptide and a DENVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide and a VEEV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide and a VEEV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide, a SINV antigenic polypeptide and a CHIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide, a SINV antigenic polypeptide and a DENV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide, a SINV antigenic polypeptide and a ZIKV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide, a SINV antigenic polypeptide and a YFV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide, a CHIKV antigenic polypeptide and a DENVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide, a CHIKV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide, a CHIKV antigenic polypeptide and a YFV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide, a DENV antigenic polypeptide and a ZIKV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide, a DENV antigenic polypeptide and a YFV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide, a ZIKV antigenic polypeptide and a YFV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a WNVantigenic polypeptide, a EEEV antigenic polypeptide and a VEEV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a WNVantigenic polypeptide, a EEEV antigenic polypeptide and a SINV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a WNVantigenic polypeptide, a EEEV antigenic polypeptide and a CHIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a WNVantigenic polypeptide, a EEEV antigenic polypeptide and a DENV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a WNVantigenic polypeptide, a EEEV antigenic polypeptide and a ZIKV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a WNVantigenic polypeptide, a EEEV antigenic polypeptide and a YFV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a WNVantigenic polypeptide, a VEEV antigenic polypeptide and a SINV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a WNVantigenic polypeptide, a VEEV antigenic polypeptide and a CHIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a WNVantigenic polypeptide, a VEEV antigenic polypeptide and a DENV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a WNVantigenic polypeptide, a VEEV antigenic polypeptide and a ZIKV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a WNVantigenic polypeptide, a VEEV antigenic polypeptide and a YFV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a WNVantigenic polypeptide, a SINV antigenic polypeptide and a CHIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a WNVantigenic polypeptide, a SINV antigenic polypeptide and a DENV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a WNVantigenic polypeptide, a SINV antigenic polypeptide and a ZIKV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a WNVantigenic polypeptide, a SINV antigenic polypeptide and YFV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a WNVantigenic polypeptide, a CHIKV antigenic polypeptide and a DENVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a WNVantigenic polypeptide, a CHIKV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a WNVantigenic polypeptide, a CHIKV antigenic polypeptide and a YFV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a WNVantigenic polypeptide, DENV antigenic polypeptide and a ZIKV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a WNVantigenic polypeptide, DENV antigenic polypeptide and a YFV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a WNVantigenic polypeptide, ZIKV antigenic polypeptide and a YFV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, EEEVantigenic polypeptide, a VEEV antigenic polypeptide and a SINV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, EEEVantigenic polypeptide, a VEEV antigenic polypeptide and a CHIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, EEEVantigenic polypeptide, a VEEV antigenic polypeptide and a DENV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, EEEVantigenic polypeptide, a VEEV antigenic polypeptide and a ZIKV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, EEEVantigenic polypeptide, a VEEV antigenic polypeptide and a YFV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a SINVantigenic polypeptide and a CHIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a SINVantigenic polypeptide and a DENV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a SINVantigenic polypeptide and a ZIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a SINVantigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a EEEVantigenic polypeptide, a CHIKV antigenic polypeptide and a DENVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a EEEVantigenic polypeptide, a CHIKV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a EEEVantigenic polypeptide, a CHIKV antigenic polypeptide and a YFV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a EEEVantigenic polypeptide, DENV antigenic polypeptide and a ZIKV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a EEEVantigenic polypeptide, DENV antigenic polypeptide and a YFV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a EEEVantigenic polypeptide, a ZIKV antigenic polypeptide and a YFV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a VEEVantigenic polypeptide, a SINV antigenic polypeptide and a CHIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a VEEVantigenic polypeptide, a SINV antigenic polypeptide and a DENV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a VEEVantigenic polypeptide, a SINV antigenic polypeptide and a ZIKV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a VEEVantigenic polypeptide, a SINV antigenic polypeptide and a YFV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a VEEVantigenic polypeptide, a CHIKV antigenic polypeptide and a DENVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a VEEVantigenic polypeptide, a CHIKV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a VEEVantigenic polypeptide, a CHIKV antigenic polypeptide and a YFV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a VEEVantigenic polypeptide, a DENV antigenic polypeptide and a ZIKV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a VEEVantigenic polypeptide, a DENV antigenic polypeptide and a YFV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a VEEVantigenic polypeptide, a ZIKV antigenic polypeptide and a YFV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a SINVantigenic polypeptide, a CHIKV antigenic polypeptide and a DENVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a SINVantigenic polypeptide, a CHIKV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a SINVantigenic polypeptide, a CHIKV antigenic polypeptide and a YFV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a SINVantigenic polypeptide, a DENV antigenic polypeptide and a ZIKV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a SINVantigenic polypeptide, a DENV antigenic polypeptide and a YFV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a SINVantigenic polypeptide, a ZIKV antigenic polypeptide and a YFV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a CHIKVantigenic polypeptide, a DENV antigenic polypeptide and a ZIKV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a CHIKVantigenic polypeptide, a DENV antigenic polypeptide and a YFV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a CHIKVantigenic polypeptide, a ZIKV antigenic polypeptide and a YFV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a DENVantigenic polypeptide, a ZIKV antigenic polypeptide and a YFV antigenicpolypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide, a EEEV antigenic polypeptide and a VEEVantigenic.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide, a EEEV antigenic polypeptide and a SINVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide, a EEEV antigenic polypeptide and a CHIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide, a EEEV antigenic polypeptide and a DENVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide, a EEEV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide, a EEEV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide, a VEEV antigenic polypeptide and a SINVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide, a VEEV antigenic polypeptide and a CHIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide, a VEEV antigenic polypeptide and a DENVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide, a VEEV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide, a VEEV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide, a VEEV antigenic polypeptide and a SINVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide, a VEEV antigenic polypeptide and a CHIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide, a VEEV antigenic polypeptide and a DENVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide, a VEEV antigenic polypeptide and a ZIKVantigenic polypeptide and a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide, a VEEV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide, a SINV antigenic polypeptide and a CHIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide, a SINV antigenic polypeptide and a DENVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide, a SINV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide, a SINV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide, a CHIKV antigenic polypeptide and a DENVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide, a CHIKV antigenic polypeptide and a ZIKVantigenic polypeptide

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide, a CHIKV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide, a DENV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide, a DENV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide, a ZIKV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aEEEV antigenic polypeptide, a VEEV antigenic polypeptide and a SINVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aEEEV antigenic polypeptide, a VEEV antigenic polypeptide and a CHIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aEEEV antigenic polypeptide, a VEEV antigenic polypeptide and a DENVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aEEEV antigenic polypeptide, a VEEV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aEEEV antigenic polypeptide, a VEEV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aEEEV antigenic polypeptide, a SINV antigenic polypeptide and a CHIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aEEEV antigenic polypeptide, a SINV antigenic polypeptide and a DENVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aEEEV antigenic polypeptide, a SINV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aEEEV antigenic polypeptide, a SINV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aEEEV antigenic polypeptide, a CHIKV antigenic polypeptide and a DENVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aEEEV antigenic polypeptide, a CHIKV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aEEEV antigenic polypeptide, a CHIKV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aEEEV antigenic polypeptide, a DENV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aEEEV antigenic polypeptide, a DENV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aEEEV antigenic polypeptide, a ZIKV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aVEEV antigenic polypeptide, a SINV antigenic polypeptide and a CHIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aVEEV antigenic polypeptide, a SINV antigenic polypeptide and a DENVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aVEEV antigenic polypeptide, a SINV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aVEEV antigenic polypeptide, a SINV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aVEEV antigenic polypeptide, a CHIKV antigenic polypeptide and a DENVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aVEEV antigenic polypeptide, a CHIKV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aVEEV antigenic polypeptide, a CHIKV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aVEEV antigenic polypeptide, a DENV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aVEEV antigenic polypeptide, a DENV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aVEEV antigenic polypeptide, a ZIKV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aSINV antigenic polypeptide, a CHIKV antigenic polypeptide and a DENVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aSINV antigenic polypeptide, a CHIKV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aSINV antigenic polypeptide, a CHIKV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aSINV antigenic polypeptide, a DENV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aSINV antigenic polypeptide, a DENV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aSINV antigenic polypeptide, a ZIKV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aCHIKV antigenic polypeptide, a DENV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aCHIKV antigenic polypeptide, a DENV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aCHIKV antigenic polypeptide, a ZIKV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aDENV antigenic polypeptide, a ZIKV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aEEEV antigenic polypeptide, a VEEV antigenic polypeptide and a SINVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aEEEV antigenic polypeptide, a VEEV antigenic polypeptide and a CHIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aEEEV antigenic polypeptide, a VEEV antigenic polypeptide and a DENVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aEEEV antigenic polypeptide, a VEEV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aEEEV antigenic polypeptide, a VEEV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aEEEV antigenic polypeptide, a SINV antigenic polypeptide and a CHIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aEEEV antigenic polypeptide, a SINV antigenic polypeptide and a DENVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aEEEV antigenic polypeptide, a SINV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aEEEV antigenic polypeptide, a SINV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aEEEV antigenic polypeptide, a CHIKV antigenic polypeptide and a DENVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aEEEV antigenic polypeptide, a CHIKV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aEEEV antigenic polypeptide, a CHIKV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aEEEV antigenic polypeptide, a DENV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aEEEV antigenic polypeptide, a DENV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aEEEV antigenic polypeptide, a ZIKV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aVEEV antigenic polypeptide, a SINV antigenic polypeptide and a CHIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aVEEV antigenic polypeptide, a SINV antigenic polypeptide and a DENVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aVEEV antigenic polypeptide, a SINV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aVEEV antigenic polypeptide, a SINV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aVEEV antigenic polypeptide, a CHIKV antigenic polypeptide and a DENVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aVEEV antigenic polypeptide, a CHIKV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aVEEV antigenic polypeptide, a CHIKV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aVEEV antigenic polypeptide, a DENV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aVEEV antigenic polypeptide, a DENV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aVEEV antigenic polypeptide, a ZIKV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aSINV antigenic polypeptide, a CHIKV antigenic polypeptide and a DENVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aSINV antigenic polypeptide, a CHIKV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aSINV antigenic polypeptide, a CHIKV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aSINV antigenic polypeptide, a DENV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aSINV antigenic polypeptide, a DENV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aSINV antigenic polypeptide, a ZIKV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aCHIKV antigenic polypeptide, a DENV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aCHIKV antigenic polypeptide, a DENV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aCHIKV antigenic polypeptide, a ZIKV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a WNV antigenic polypeptide, aDENV antigenic polypeptide, a ZIKV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aVEEV antigenic polypeptide, a SINV antigenic polypeptide and a CHIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aVEEV antigenic polypeptide, a SINV antigenic polypeptide and a DENVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aVEEV antigenic polypeptide, a SINV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aVEEV antigenic polypeptide, a SINV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aVEEV antigenic polypeptide, a CHIKV antigenic polypeptide and a DENVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aVEEV antigenic polypeptide, a CHIKV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aVEEV antigenic polypeptide, a CHIKV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aVEEV antigenic polypeptide, a DENV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aVEEV antigenic polypeptide, a DENV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aVEEV antigenic polypeptide, a ZIKV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aSINV antigenic polypeptide, a CHIKV antigenic polypeptide and a DENVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aSINV antigenic polypeptide, a CHIKV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aSINV antigenic polypeptide, a CHIKV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aSINV antigenic polypeptide, a DENV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aSINV antigenic polypeptide, a DENV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aSINV antigenic polypeptide, a ZIKV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aSINV antigenic polypeptide, a DENV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aSINV antigenic polypeptide, a DENV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a EEEV antigenic polypeptide, aSINV antigenic polypeptide, a ZIKV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding VEEV antigenic polypeptide, aSINV antigenic polypeptide, a CHIKV antigenic polypeptide and a DENVantigenic polypeptide

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding VEEV antigenic polypeptide, aSINV antigenic polypeptide, a CHIKV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding VEEV antigenic polypeptide, aSINV antigenic polypeptide, a CHIKV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding VEEV antigenic polypeptide, aSINV antigenic polypeptide, a DENV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding VEEV antigenic polypeptide, aSINV antigenic polypeptide, a DENV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding VEEV antigenic polypeptide, aSINV antigenic polypeptide, a ZIKV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding VEEV antigenic polypeptide, aCHIKV antigenic polypeptide, a DENV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding VEEV antigenic polypeptide, aCHIKV antigenic polypeptide, a DENV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding VEEV antigenic polypeptide, aCHIKV antigenic polypeptide, a ZIKV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding VEEV antigenic polypeptide, aDENV antigenic polypeptide, a ZIKV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding SINV antigenic polypeptide, aCHIKV antigenic polypeptide, a DENV antigenic polypeptide and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding SINV antigenic polypeptide, aCHIKV antigenic polypeptide, a DENV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding SINV antigenic polypeptide, aCHIKV antigenic polypeptide, a ZIKV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding SINV antigenic polypeptide, aDENV antigenic polypeptide, a ZIKV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding CHIKV antigenic polypeptide, aDENV antigenic polypeptide, a ZIKV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide, a EEEV antigenic polypeptide, a VEEVantigenic polypeptide, a SINV antigenic polypeptide, a CHIKV antigenicpolypeptide, a DENV antigenic polypeptide, a ZIKV antigenic polypeptideand a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a WNVantigenic polypeptide, a EEEV antigenic polypeptide, a VEEV antigenicpolypeptide, a SINV antigenic polypeptide, a CHIKV antigenicpolypeptide, a DENV antigenic polypeptide, a ZIKV antigenic polypeptideand a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide, a EEEV antigenic polypeptide, a VEEV antigenicpolypeptide, a SINV antigenic polypeptide, a CHIKV antigenicpolypeptide, a DENV antigenic polypeptide, a ZIKV antigenic polypeptideand a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide, a WNV antigenic polypeptide, a VEEV antigenicpolypeptide, a SINV antigenic polypeptide, a CHIKV antigenicpolypeptide, a DENV antigenic polypeptide, a ZIKV antigenic polypeptideand a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide, a WNV antigenic polypeptide, a EEEV antigenicpolypeptide, a SINV antigenic polypeptide, a CHIKV antigenicpolypeptide, a DENV antigenic polypeptide, a ZIKV antigenic polypeptideand a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide, a WNV antigenic polypeptide, a EEEV antigenicpolypeptide, a VEEV antigenic polypeptide, a CHIKV antigenicpolypeptide, a DENV antigenic polypeptide, a ZIKV antigenic polypeptideand a YFV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide, a WNV antigenic polypeptide, a EEEV antigenicpolypeptide, a VEEV antigenic polypeptide, a SINV antigenic polypeptide,a DENV antigenic polypeptide, a ZIKV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide, a WNV antigenic polypeptide, a EEEV antigenicpolypeptide, a VEEV antigenic polypeptide, a SINV antigenic polypeptide,a CHIKV antigenic polypeptide, a ZIKV antigenic polypeptide and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide, a WNV antigenic polypeptide, a EEEV antigenicpolypeptide, a VEEV antigenic polypeptide, a SINV antigenic polypeptide,a CHIKV antigenic polypeptide, a DENV antigenic polypeptide, and a YFVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide, a WNV antigenic polypeptide, a EEEV antigenicpolypeptide, a VEEV antigenic polypeptide, a SINV antigenic polypeptide,a CHIKV antigenic polypeptide, a DENV antigenic polypeptide, and a ZIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide, a EEEV antigenic polypeptide, a VEEVantigenic polypeptide, and a SINV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide, a EEEV antigenic polypeptide, a VEEVantigenic polypeptide, a SINV antigenic polypeptide, and a CHIKVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide, a EEEV antigenic polypeptide, a SINVantigenic polypeptide, and a CHIKV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a JEV antigenic polypeptide, aWNV antigenic polypeptide, a EEEV antigenic polypeptide, a SINVantigenic polypeptide, a CHIKV antigenic polypeptide and a DENVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises atleast two RNA (e.g., mRNA) polynucleotides selected from Malaria (e.g.,P. falciparum, P. vivax, P. malariae and/or P. ovale) antigenicpolypeptides, JEV antigenic polypeptides, WNV antigenic polypeptides,EEEV antigenic polypeptides, VEEV antigenic polypeptides, SINV antigenicpolypeptides, CHIKV antigenic polypeptides, DENV antigenic polypeptides,ZIKV antigenic polypeptides and a YFV antigenic polypeptides.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises atleast three RNA (e.g., mRNA) polynucleotides selected from Malaria(e.g., P. falciparum, P. vivax, P. malariae and/or P. ovale) antigenicpolypeptides, JEV antigenic polypeptides, WNV antigenic polypeptides,EEEV antigenic polypeptides, VEEV antigenic polypeptides, SINV antigenicpolypeptides, CHIKV antigenic polypeptides, DENV antigenic polypeptides,ZIKV antigenic polypeptides and a YFV antigenic polypeptides.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises atleast four RNA (e.g., mRNA) polynucleotides selected from Malaria (e.g.,P. falciparum, P. vivax, P. malariae and/or P. ovale) antigenicpolypeptides, JEV antigenic polypeptides, WNV antigenic polypeptides,EEEV antigenic polypeptides, VEEV antigenic polypeptides, SINV antigenicpolypeptides, CHIKV antigenic polypeptides, DENV antigenic polypeptides,ZIKV antigenic polypeptides and a YFV antigenic polypeptides.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises atleast five RNA (e.g., mRNA) polynucleotides selected from Malaria (e.g.,P. falciparum, P. vivax, P. malariae and/or P. ovale) antigenicpolypeptides, JEV antigenic polypeptides, WNV antigenic polypeptides,EEEV antigenic polypeptides, VEEV antigenic polypeptides, SINV antigenicpolypeptides, CHIKV antigenic polypeptides, DENV antigenic polypeptides,ZIKV antigenic polypeptides and a YFV antigenic polypeptides.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises atleast six RNA (e.g., mRNA) polynucleotides selected from Malaria (e.g.,P. falciparum, P. vivax, P. malariae and/or P. ovale) antigenicpolypeptides, JEV antigenic polypeptides, WNV antigenic polypeptides,EEEV antigenic polypeptides, VEEV antigenic polypeptides, SINV antigenicpolypeptides, CHIKV antigenic polypeptides, DENV antigenic polypeptides,ZIKV antigenic polypeptides and a YFV antigenic polypeptides.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises atleast seven RNA (e.g., mRNA) polynucleotides selected from Malaria(e.g., P. falciparum, P. vivax, P. malariae and/or P. ovale) antigenicpolypeptides, JEV antigenic polypeptides, WNV antigenic polypeptides,EEEV antigenic polypeptides, VEEV antigenic polypeptides, SINV antigenicpolypeptides, CHIKV antigenic polypeptides, DENV antigenic polypeptides,ZIKV antigenic polypeptides and a YFV antigenic polypeptides.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises atleast eight RNA (e.g., mRNA) polynucleotides selected from Malaria(e.g., P. falciparum, P. vivax, P. malariae and/or P. ovale) antigenicpolypeptides, JEV antigenic polypeptides, WNV antigenic polypeptides,EEEV antigenic polypeptides, VEEV antigenic polypeptides, SINV antigenicpolypeptides, CHIKV antigenic polypeptides, DENV antigenic polypeptides,ZIKV antigenic polypeptides and a YFV antigenic polypeptides.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises atleast nine RNA (e.g., mRNA) polynucleotides selected from Malaria (e.g.,P. falciparum, P. vivax, P. malariae and/or P. ovale) antigenicpolypeptides, JEV antigenic polypeptides, WNV antigenic polypeptides,EEEV antigenic polypeptides, VEEV antigenic polypeptides, SINV antigenicpolypeptides, CHIKV antigenic polypeptides, DENV antigenic polypeptides,ZIKV antigenic polypeptides and a YFV antigenic polypeptides.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises atleast ten RNA (e.g., mRNA) polynucleotides selected from Malaria (e.g.,P. falciparum, P. vivax, P. malariae and/or P. ovale) antigenicpolypeptides, JEV antigenic polypeptides, WNV antigenic polypeptides,EEEV antigenic polypeptides, VEEV antigenic polypeptides, SINV antigenicpolypeptides, CHIKV antigenic polypeptides, DENV antigenic polypeptides,ZIKV antigenic polypeptides and a YFV antigenic polypeptides.

Additional combination vaccines are encompassed by the followingnumbered paragraphs:

1. A combination vaccine comprising at least one RNA (e.g., mRNA)encoding at least one tropical disease antigenic polypeptide.

2. The combination vaccine of paragraph 1, wherein the at least onepolypeptide is at least one Malaria (e.g., P. falciparum, P. vivax, P.malariae and/or P. ovale) antigenic polypeptide.

3. The combination vaccine of paragraph 1 or 2, wherein the at least onepolypeptide is at least one JEV antigenic polypeptide.

4. The combination vaccine of any one of paragraphs 1-3, wherein the atleast one polypeptide is at least one WNV antigenic polypeptide.

5. The combination vaccine of any one of paragraphs 1-4, wherein the atleast one polypeptide is at least one EEEV antigenic polypeptide.

6. The combination vaccine of any one of paragraphs 1-5, wherein the atleast one polypeptide is at least one VEEV antigenic polypeptide.

7. The combination vaccine of any one of paragraphs 1-6, wherein the atleast one polypeptide is at least one SINV antigenic polypeptide.

8. The combination vaccine of any one of paragraphs 1-7, wherein the atleast one polypeptide is at least one CHIKV antigenic polypeptide.

9. The combination vaccine of any one of paragraphs 1-8, wherein the atleast one polypeptide is at least one DENV antigenic polypeptide.

10. The combination vaccine of any one of paragraphs 1-9, wherein the atleast one polypeptide is at least one ZIKV antigenic polypeptide.

11. The combination vaccine of any one of paragraphs 1-10, wherein theat least one polypeptide is at least one YFV antigenic polypeptide.

It has been discovered that the mRNA vaccines described herein aresuperior to current vaccines in several ways. First, the lipidnanoparticle (LNP) delivery is superior to other formulations includinga protamine base approach described in the literature and no additionaladjuvants are to be necessary. The use of LNPs enables the effectivedelivery of chemically modified or unmodified mRNA vaccines.Additionally it has been demonstrated herein that both modified andunmodified LNP formulated mRNA vaccines were superior to conventionalvaccines by a significant degree. In some embodiments the mRNA vaccinesof the invention are superior to conventional vaccines by a factor of atleast 10 fold, 20 fold, 40 fold, 50 fold, 100 fold, 500 fold or 1,000fold.

Although attempts have been made to produce functional RNA vaccines,including mRNA vaccines and self-replicating RNA vaccines, thetherapeutic efficacy of these RNA vaccines has not yet been fullyestablished. Quite surprisingly, the inventors have discovered,according to aspects of the invention a class of formulations fordelivering mRNA vaccines in vivo that results in significantly enhanced,and in many respects synergistic, immune responses including enhancedantigen generation and functional antibody production withneutralization capability. These results can be achieved even whensignificantly lower doses of the mRNA are administered in comparisonwith mRNA doses used in other classes of lipid based formulations. Theformulations of the invention have demonstrated significant unexpectedin vivo immune responses sufficient to establish the efficacy offunctional mRNA vaccines as prophylactic and therapeutic agents.Additionally, self-replicating RNA vaccines rely on viral replicationpathways to deliver enough RNA to a cell to produce an immunogenicresponse. The formulations of the invention do not require viralreplication to produce enough protein to result in a strong immuneresponse. Thus, the mRNA of the invention are not self-replicating RNAand do not include components necessary for viral replication.

The invention involves, in some aspects, the surprising finding thatlipid nanoparticle (LNP) formulations significantly enhance theeffectiveness of mRNA vaccines, including chemically modified andunmodified mRNA vaccines. The efficacy of mRNA vaccines formulated inLNP was examined in vivo using several distinct antigens. The resultspresented herein demonstrate the unexpected superior efficacy of themRNA vaccines formulated in LNP over other commercially availablevaccines.

In addition to providing an enhanced immune response, the formulationsof the invention generate a more rapid immune response with fewer dosesof antigen than other vaccines tested. The mRNA-LNP formulations of theinvention also produce quantitatively and qualitatively better immuneresponses than vaccines formulated in a different carriers.

The data described herein demonstrate that the formulations of theinvention produced significant unexpected improvements over existingantigen vaccines. Additionally, the mRNA-LNP formulations of theinvention are superior to other vaccines even when the dose of mRNA islower than other vaccines.

The LNP used in the studies described herein has been used previously todeliver siRNA in various animal models as well as in humans. In view ofthe observations made in association with the siRNA delivery of LNPformulations, the fact that LNP is useful in vaccines is quitesurprising. It has been observed that therapeutic delivery of siRNAformulated in LNP causes an undesirable inflammatory response associatedwith a transient IgM response, typically leading to a reduction inantigen production and a compromised immune response. In contrast to thefindings observed with siRNA, the LNP-mRNA formulations of the inventionare demonstrated herein to generate enhanced IgG levels, sufficient forprophylactic and therapeutic methods rather than transient IgMresponses.

Nucleic Acids/Polynucleotides

Tropical disease vaccines, as provided herein, comprise at least one(one or more) ribonucleic acid (RNA) (e.g., mRNA) polynucleotide havingan open reading frame encoding at least one Malaria (e.g., P.falciparum, P. vivax, P. malariae and/or P. ovale), JEV, WNV, EEEV,VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV antigenic polypeptide. The term“nucleic acid” includes any compound and/or substance that comprises apolymer of nucleotides (nucleotide monomer). These polymers are referredto as polynucleotides. Thus, the terms “nucleic acid” and“polynucleotide” are used interchangeably.

Nucleic acids may be or may include, for example, ribonucleic acids(RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs),glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), lockednucleic acids (LNAs, including LNA having a β-D-ribo configuration,α-LNA having an α-L-ribo configuration (a diastereomer of LNA),2′-amino-LNA having a 2′-amino functionalization, and 2′-amino-α-LNAhaving a 2′-amino functionalization), ethylene nucleic acids (ENA),cyclohexenyl nucleic acids (CeNA) or chimeras or combinations thereof.

In some embodiments, polynucleotides of the present disclosure functionas messenger RNA (mRNA). “Messenger RNA” (mRNA) refers to anypolynucleotide that encodes a (at least one) polypeptide (anaturally-occurring, non-naturally-occurring, or modified polymer ofamino acids) and can be translated to produce the encoded polypeptide invitro, in vivo, in situ or ex vivo. The skilled artisan will appreciatethat, except where otherwise noted, polynucleotide sequences set forthin the instant application will recite “T”s in a representative DNAsequence but where the sequence represents RNA (e.g., mRNA), the “T”swould be substituted for “U”s. Thus, any of the RNA polynucleotidesencoded by a DNA identified by a particular sequence identificationnumber may also comprise the corresponding RNA (e.g., mRNA) sequenceencoded by the DNA, where each “T” of the DNA sequence is substitutedwith “U.”

The basic components of an mRNA molecule typically include at least onecoding region, a 5′ untranslated region (UTR), a 3′ UTR, a 5′ cap and apoly-A tail. Polynucleotides of the present disclosure may function asmRNA but can be distinguished from wild-type mRNA in their functionaland/or structural design features, which serve to overcome existingproblems of effective polypeptide expression using nucleic-acid basedtherapeutics.

In some embodiments, a RNA polynucleotide of an RNA (e.g., mRNA) vaccineencodes 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7,3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6,6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9 or 9-10 antigenicpolypeptides. In some embodiments, a RNA (e.g., mRNA) polynucleotide ofa tropical disease vaccine encodes at least 10, 20, 30, 40, 50, 60, 70,80, 90 or 100 antigenic polypeptides. In some embodiments, a RNA (e.g.,mRNA) polynucleotide of a tropical disease vaccine encodes at least 100or at least 200 antigenic polypeptides. In some embodiments, a RNApolynucleotide of a tropical disease vaccine encodes 1-10, 5-15, 10-20,15-25, 20-30, 25-35, 30-40, 35-45, 40-50, 1-50, 1-100, 2-50 or 2-100antigenic polypeptides.

Polynucleotides of the present disclosure, in some embodiments, arecodon optimized. Codon optimization methods are known in the art and maybe used as provided herein. Codon optimization, in some embodiments, maybe used to match codon frequencies in target and host organisms toensure proper folding; bias GC content to increase mRNA stability orreduce secondary structures; minimize tandem repeat codons or base runsthat may impair gene construction or expression; customizetranscriptional and translational control regions; insert or removeprotein trafficking sequences; remove/add post translation modificationsites in encoded protein (e.g. glycosylation sites); add, remove orshuffle protein domains; insert or delete restriction sites; modifyribosome binding sites and mRNA degradation sites; adjust translationalrates to allow the various domains of the protein to fold properly; orto reduce or eliminate problem secondary structures within thepolynucleotide. Codon optimization tools, algorithms and services areknown in the art—non-limiting examples include services from GeneArt(Life Technologies), DNA2.0 (Menlo Park Calif.) and/or proprietarymethods. In some embodiments, the open reading frame (ORF) sequence isoptimized using optimization algorithms.

In some embodiments, a codon optimized sequence shares less than 95%sequence identity, less than 90% sequence identity, less than 85%sequence identity, less than 80% sequence identity, or less than 75%sequence identity to a naturally-occurring or wild-type sequence (e.g.,a naturally-occurring or wild-type mRNA sequence encoding a polypeptideor protein of interest (e.g., an antigenic protein or antigenicpolypeptide)).

In some embodiments, a codon-optimized sequence shares between 65% and85% (e.g., between about 67% and about 85%, or between about 67% andabout 80%) sequence identity to a naturally-occurring sequence or awild-type sequence (e.g., a naturally-occurring or wild-type mRNAsequence encoding a polypeptide or protein of interest (e.g., anantigenic protein or polypeptide)). In some embodiments, acodon-optimized sequence shares between 65% and 75%, or about 80%sequence identity to a naturally-occurring sequence or wild-typesequence (e.g., a naturally-occurring or wild-type mRNA sequenceencoding a polypeptide or protein of interest (e.g., an antigenicprotein or polypeptide)).

In some embodiments a codon-optimized RNA (e.g., mRNA) may, forinstance, be one in which the levels of G/C are enhanced. TheG/C-content of nucleic acid molecules may influence the stability of theRNA. RNA having an increased amount of guanine (G) and/or cytosine (C)residues may be functionally more stable than nucleic acids containing alarge amount of adenine (A) and thymine (T) or uracil (U) nucleotides.WO02/098443 discloses a pharmaceutical composition containing an mRNAstabilized by sequence modifications in the translated region. Due tothe degeneracy of the genetic code, the modifications work bysubstituting existing codons for those that promote greater RNAstability without changing the resulting amino acid. The approach islimited to coding regions of the RNA.

Antigens/Antigenic Polypeptides

In some embodiments, an antigenic polypeptide (e.g., at least oneMalaria (e.g., P. falciparum, P. vivax, P. malariae and/or P. ovale),JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV antigenicpolypeptide) is longer than 25 amino acids and shorter than 50 aminoacids. Polypeptides include gene products, naturally occurringpolypeptides, synthetic polypeptides, homologs, orthologs, paralogs,fragments and other equivalents, variants, and analogs of the foregoing.A polypeptide may be a single molecule or may be a multi-molecularcomplex such as a dimer, trimer or tetramer. Polypeptides may alsocomprise single chain polypeptides or multichain polypeptides, such asantibodies or insulin, and may be associated or linked to each other.Most commonly, disulfide linkages are found in multichain polypeptides.The term “polypeptide” may also apply to amino acid polymers in which atleast one amino acid residue is an artificial chemical analogue of acorresponding naturally-occurring amino acid.

A “polypeptide variant” is a molecule that differs in its amino acidsequence relative to a native sequence or a reference sequence. Aminoacid sequence variants may possess substitutions, deletions, insertions,or a combination of any two or three of the foregoing, at certainpositions within the amino acid sequence, as compared to a nativesequence or a reference sequence. Ordinarily, variants possess at least50% identity to a native sequence or a reference sequence. In someembodiments, variants share at least 80% identity or at least 90%identity with a native sequence or a reference sequence.

In some embodiments “variant mimics” are provided. A “variant mimic”contains at least one amino acid that would mimic an activated sequence.For example, glutamate may serve as a mimic for phosphoro-threonineand/or phosphoro-serine. Alternatively, variant mimics may result indeactivation or in an inactivated product containing the mimic. Forexample, phenylalanine may act as an inactivating substitution fortyrosine, or alanine may act as an inactivating substitution for serine.

“Orthologs” refers to genes in different species that evolved from acommon ancestral gene by speciation. Normally, orthologs retain the samefunction in the course of evolution. Identification of orthologs isimportant for reliable prediction of gene function in newly sequencedgenomes.

“Analogs” is meant to include polypeptide variants that differ by one ormore amino acid alterations, for example, substitutions, additions ordeletions of amino acid residues that still maintain one or more of theproperties of the parent or starting polypeptide.

The present disclosure provides several types of compositions that arepolynucleotide or polypeptide based, including variants and derivatives.These include, for example, substitutional, insertional, deletion andcovalent variants and derivatives. The term “derivative” is synonymouswith the term “variant” and generally refers to a molecule that has beenmodified and/or changed in any way relative to a reference molecule or astarting molecule.

As such, polynucleotides encoding peptides or polypeptides containingsubstitutions, insertions and/or additions, deletions and covalentmodifications with respect to reference sequences, in particular thepolypeptide sequences disclosed herein, are included within the scope ofthis disclosure. For example, sequence tags or amino acids, such as oneor more lysines, can be added to peptide sequences (e.g., at theN-terminal or C-terminal ends). Sequence tags can be used for peptidedetection, purification or localization. Lysines can be used to increasepeptide solubility or to allow for biotinylation. Alternatively, aminoacid residues located at the carboxy and amino terminal regions of theamino acid sequence of a peptide or protein may optionally be deletedproviding for truncated sequences. Certain amino acids (e.g., C-terminalresidues or N-terminal residues) alternatively may be deleted dependingon the use of the sequence, as for example, expression of the sequenceas part of a larger sequence that is soluble, or linked to a solidsupport.

“Substitutional variants” when referring to polypeptides are those thathave at least one amino acid residue in a native or starting sequenceremoved and a different amino acid inserted in its place at the sameposition. Substitutions may be single, where only one amino acid in themolecule has been substituted, or they may be multiple, where two ormore (e.g., 3, 4 or 5) amino acids have been substituted in the samemolecule.

As used herein the term “conservative amino acid substitution” refers tothe substitution of an amino acid that is normally present in thesequence with a different amino acid of similar size, charge, orpolarity. Examples of conservative substitutions include thesubstitution of a non-polar (hydrophobic) residue such as isoleucine,valine and leucine for another non-polar residue. Likewise, examples ofconservative substitutions include the substitution of one polar(hydrophilic) residue for another such as between arginine and lysine,between glutamine and asparagine, and between glycine and serine.Additionally, the substitution of a basic residue such as lysine,arginine or histidine for another, or the substitution of one acidicresidue such as aspartic acid or glutamic acid for another acidicresidue are additional examples of conservative substitutions. Examplesof non-conservative substitutions include the substitution of anon-polar (hydrophobic) amino acid residue such as isoleucine, valine,leucine, alanine, methionine for a polar (hydrophilic) residue such ascysteine, glutamine, glutamic acid or lysine and/or a polar residue fora non-polar residue.

“Features” when referring to polypeptide or polynucleotide are definedas distinct amino acid sequence-based or nucleotide-based components ofa molecule respectively. Features of the polypeptides encoded by thepolynucleotides include surface manifestations, local conformationalshape, folds, loops, half-loops, domains, half-domains, sites, terminiand any combination(s) thereof.

As used herein when referring to polypeptides the term “domain” refersto a motif of a polypeptide having one or more identifiable structuralor functional characteristics or properties (e.g., binding capacity,serving as a site for protein-protein interactions).

As used herein when referring to polypeptides the terms “site” as itpertains to amino acid based embodiments is used synonymously with“amino acid residue” and “amino acid side chain.” As used herein whenreferring to polynucleotides the terms “site” as it pertains tonucleotide based embodiments is used synonymously with “nucleotide.” Asite represents a position within a peptide or polypeptide orpolynucleotide that may be modified, manipulated, altered, derivatizedor varied within the polypeptide-based or polynucleotide-basedmolecules.

As used herein the terms “termini” or “terminus” when referring topolypeptides or polynucleotides refer to an extremity of a polypeptideor polynucleotide respectively. Such extremity is not limited only tothe first or final site of the polypeptide or polynucleotide but mayinclude additional amino acids or nucleotides in the terminal regions.Polypeptide-based molecules may be characterized as having both anN-terminus (terminated by an amino acid with a free amino group (NH₂))and a C-terminus (terminated by an amino acid with a free carboxyl group(COOH)). Proteins are in some cases made up of multiple polypeptidechains brought together by disulfide bonds or by non-covalent forces(multimers, oligomers). These proteins have multiple N- and C-termini.Alternatively, the termini of the polypeptides may be modified such thatthey begin or end, as the case may be, with a non-polypeptide basedmoiety such as an organic conjugate.

As recognized by those skilled in the art, protein fragments, functionalprotein domains, and homologous proteins are also considered to bewithin the scope of polypeptides of interest. For example, providedherein is any protein fragment (meaning a polypeptide sequence at leastone amino acid residue shorter than a reference polypeptide sequence butotherwise identical) of a reference protein having a length of 10, 20,30, 40, 50, 60, 70, 80, 90, 100 or longer than 100 amino acids. Inanother example, any protein that includes a stretch of 20, 30, 40, 50,or 100 (contiguous) amino acids that are 40%, 50%, 60%, 70%, 80%, 90%,95%, or 100% identical to any of the sequences described herein can beutilized in accordance with the disclosure. In some embodiments, apolypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations asshown in any of the sequences provided herein or referenced herein. Inanother example, any protein that includes a stretch of 20, 30, 40, 50,or 100 amino acids that are greater than 80%, 90%, 95%, or 100%identical to any of the sequences described herein, wherein the proteinhas a stretch of 5, 10, 15, 20, 25, or 30 amino acids that are less than80%, 75%, 70%, 65% to 60% identical to any of the sequences describedherein can be utilized in accordance with the disclosure.

Polypeptide or polynucleotide molecules of the present disclosure mayshare a certain degree of sequence similarity or identity with thereference molecules (e.g., reference polypeptides or referencepolynucleotides), for example, with art-described molecules (e.g.,engineered or designed molecules or wild-type molecules). The term“identity,” as known in the art, refers to a relationship between thesequences of two or more polypeptides or polynucleotides, as determinedby comparing the sequences. In the art, identity also means the degreeof sequence relatedness between two sequences as determined by thenumber of matches between strings of two or more amino acid residues ornucleic acid residues. Identity measures the percent of identicalmatches between the smaller of two or more sequences with gap alignments(if any) addressed by a particular mathematical model or computerprogram (e.g., “algorithms”). Identity of related peptides can bereadily calculated by known methods. “% identity” as it applies topolypeptide or polynucleotide sequences is defined as the percentage ofresidues (amino acid residues or nucleic acid residues) in the candidateamino acid or nucleic acid sequence that are identical with the residuesin the amino acid sequence or nucleic acid sequence of a second sequenceafter aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent identity. Methods and computer programs forthe alignment are well known in the art. Identity depends on acalculation of percent identity but may differ in value due to gaps andpenalties introduced in the calculation. Generally, variants of aparticular polynucleotide or polypeptide have at least 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% but less than 100% sequence identity to that particularreference polynucleotide or polypeptide as determined by sequencealignment programs and parameters described herein and known to thoseskilled in the art. Such tools for alignment include those of the BLASTsuite (Stephen F. Altschul, et al. (1997).“ Gapped BLAST and PSI-BLAST:a new generation of protein database search programs,” Nucleic AcidsRes. 25:3389-3402). Another popular local alignment technique is basedon the Smith-Waterman algorithm (Smith, T. F. & Waterman, M. S. (1981)“Identification of common molecular subsequences.” J. Mol. Biol.147:195-197). A general global alignment technique based on dynamicprogramming is the Needleman-Wunsch algorithm (Needleman, S. B. &Wunsch, C. D. (1970) “A general method applicable to the search forsimilarities in the amino acid sequences of two proteins.” J. Mol. Biol.48:443-453). More recently, a Fast Optimal Global Sequence AlignmentAlgorithm (FOGSAA) was developed that purportedly produces globalalignment of nucleotide and protein sequences faster than other optimalglobal alignment methods, including the Needleman-Wunsch algorithm.Other tools are described herein, specifically in the definition of“identity” below.

As used herein, the term “homology” refers to the overall relatednessbetween polymeric molecules, e.g. between nucleic acid molecules (e.g.DNA molecules and/or RNA molecules) and/or between polypeptidemolecules. Polymeric molecules (e.g. nucleic acid molecules (e.g. DNAmolecules and/or RNA molecules) and/or polypeptide molecules) that sharea threshold level of similarity or identity determined by alignment ofmatching residues are termed homologous. Homology is a qualitative termthat describes a relationship between molecules and can be based uponthe quantitative similarity or identity. Similarity or identity is aquantitative term that defines the degree of sequence match between twocompared sequences. In some embodiments, polymeric molecules areconsidered to be “homologous” to one another if their sequences are atleast 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 99% identical or similar. The term “homologous” necessarilyrefers to a comparison between at least two sequences (polynucleotide orpolypeptide sequences). Two polynucleotide sequences are consideredhomologous if the polypeptides they encode are at least 50%, 60%, 70%,80%, 90%, 95%, or even 99% for at least one stretch of at least 20 aminoacids. In some embodiments, homologous polynucleotide sequences arecharacterized by the ability to encode a stretch of at least 4-5uniquely specified amino acids. For polynucleotide sequences less than60 nucleotides in length, homology is determined by the ability toencode a stretch of at least 4-5 uniquely specified amino acids. Twoprotein sequences are considered homologous if the proteins are at least50%, 60%, 70%, 80%, or 90% identical for at least one stretch of atleast 20 amino acids.

Homology implies that the compared sequences diverged in evolution froma common origin. The term “homolog” refers to a first amino acidsequence or nucleic acid sequence (e.g., gene (DNA or RNA) or proteinsequence) that is related to a second amino acid sequence or nucleicacid sequence by descent from a common ancestral sequence. The term“homolog” may apply to the relationship between genes and/or proteinsseparated by the event of speciation or to the relationship betweengenes and/or proteins separated by the event of genetic duplication.“Orthologs” are genes (or proteins) in different species that evolvedfrom a common ancestral gene (or protein) by speciation. Typically,orthologs retain the same function in the course of evolution.“Paralogs” are genes (or proteins) related by duplication within agenome. Orthologs retain the same function in the course of evolution,whereas paralogs evolve new functions, even if these are related to theoriginal one.

The term “identity” refers to the overall relatedness between polymericmolecules, for example, between polynucleotide molecules (e.g. DNAmolecules and/or RNA molecules) and/or between polypeptide molecules.Calculation of the percent identity of two polynucleic acid sequences,for example, can be performed by aligning the two sequences for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second nucleic acid sequences for optimal alignment andnon-identical sequences can be disregarded for comparison purposes). Incertain embodiments, the length of a sequence aligned for comparisonpurposes is at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, or 100% of thelength of the reference sequence. The nucleotides at correspondingnucleotide positions are then compared. When a position in the firstsequence is occupied by the same nucleotide as the correspondingposition in the second sequence, then the molecules are identical atthat position. The percent identity between the two sequences is afunction of the number of identical positions shared by the sequences,taking into account the number of gaps, and the length of each gap,which needs to be introduced for optimal alignment of the two sequences.The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. For example, the percent identity between two nucleic acidsequences can be determined using methods such as those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991;each of which is incorporated herein by reference. For example, thepercent identity between two nucleic acid sequences can be determinedusing the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), whichhas been incorporated into the ALIGN program (version 2.0) using aPAM120 weight residue table, a gap length penalty of 12 and a gappenalty of 4. The percent identity between two nucleic acid sequencescan, alternatively, be determined using the GAP program in the GCGsoftware package using an NWSgapdna.CMP matrix. Methods commonlyemployed to determine percent identity between sequences include, butare not limited to those disclosed in Carillo, H., and Lipman, D., SIAMJ Applied Math., 48:1073 (1988); incorporated herein by reference.Techniques for determining identity are codified in publicly availablecomputer programs. Exemplary computer software to determine homologybetween two sequences include, but are not limited to, GCG programpackage, Devereux, J., et al., Nucleic Acids Research, 12, 387 (1984)),BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec. Biol., 215,403 (1990)).

Multiprotein and Multicomponent Vaccines

The present disclosure encompasses tropical disease vaccines comprisingmultiple RNA (e.g., mRNA) polynucleotides, each encoding a singleantigenic polypeptide, as well as tropical disease vaccines comprising asingle RNA polynucleotide encoding more than one antigenic polypeptide(e.g., as a fusion polypeptide). Thus, a vaccine composition comprisinga RNA (e.g., mRNA) polynucleotide having an open reading frame encodinga first antigenic polypeptide and a RNA (e.g., mRNA) polynucleotidehaving an open reading frame encoding a second antigenic polypeptideencompasses (a) vaccines that comprise a first RNA polynucleotideencoding a first antigenic polypeptide and a second RNA polynucleotideencoding a second antigenic polypeptide, and (b) vaccines that comprisea single RNA polynucleotide encoding a first and second antigenicpolypeptide (e.g., as a fusion polypeptide). RNA (e.g., mRNA) vaccinesof the present disclosure, in some embodiments, comprise 2-10 (e.g., 2,3, 4, 5, 6, 7, 8, 9 or 10), or more, RNA polynucleotides having an openreading frame, each of which encodes a different antigenic polypeptide(or a single RNA polynucleotide encoding 2-10, or more, differentantigenic polypeptides). The antigenic polypeptides may be selected fromMalaria (e.g., P. falciparum, P. vivax, P. malariae and/or P. ovale),JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and YFV antigenicpolypeptides.

In some embodiments, a tropical disease vaccine comprises a RNA (e.g.,mRNA) polynucleotide having an open reading frame encoding a viralcapsid protein, a RNA (e.g., mRNA) polynucleotide having an open readingframe encoding a viral premembrane/membrane protein, and a RNA (e.g.,mRNA) polynucleotide having an open reading frame encoding a viralenvelope protein. In some embodiments, a tropical disease vaccinecomprises a RNA (e.g., mRNA) polynucleotide having an open reading frameencoding a viral fusion (F) protein and a RNA polynucleotide having anopen reading frame encoding a viral major surface glycoprotein (Gprotein). In some embodiments, a vaccine comprises a RNA (e.g., mRNA)polynucleotide having an open reading frame encoding a viral F protein.In some embodiments, a vaccine comprises a RNA (e.g., mRNA)polynucleotide having an open reading frame encoding a viral G protein.In some embodiments, a vaccine comprises a RNA (e.g., mRNA)polynucleotide having an open reading frame encoding a HN protein.

In some embodiments, a multicomponent vaccine comprises at least one RNA(e.g., mRNA) polynucleotide encoding at least one antigenic polypeptidefused to a signal peptide (e.g., SEQ ID NO: 304-307). The signal peptidemay be fused at the N-terminus or the C-terminus of an antigenicpolypeptide. An antigenic polypeptide fused to a signal peptide may beselected from Malaria (e.g., P. falciparum, P. vivax, P. malariae and/orP. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and YFVantigenic polypeptides.

Signal Peptides

In some embodiments, antigenic polypeptides encoded by tropical diseaseRNA (e.g., mRNA) polynucleotides comprise a signal peptide. Signalpeptides, comprising the N-terminal 15-60 amino acids of proteins, aretypically needed for the translocation across the membrane on thesecretory pathway and, thus, universally control the entry of mostproteins both in eukaryotes and prokaryotes to the secretory pathway.Signal peptides generally include three regions: an N-terminal region ofdiffering length, which usually comprises positively charged aminoacids; a hydrophobic region; and a short carboxy-terminal peptideregion. In eukaryotes, the signal peptide of a nascent precursor protein(pre-protein) directs the ribosome to the rough endoplasmic reticulum(ER) membrane and initiates the transport of the growing peptide chainacross it for processing. ER processing produces mature proteins,wherein the signal peptide is cleaved from precursor proteins, typicallyby a ER-resident signal peptidase of the host cell, or they remainuncleaved and function as a membrane anchor. A signal peptide may alsofacilitate the targeting of the protein to the cell membrane. The signalpeptide, however, is not responsible for the final destination of themature protein. Secretory proteins devoid of additional address tags intheir sequence are by default secreted to the external environment.During recent years, a more advanced view of signal peptides hasevolved, showing that the functions and immunodominance of certainsignal peptides are much more versatile than previously anticipated.

Tropical disease vaccines of the present disclosure may comprise, forexample, RNA (e.g., mRNA) polynucleotides encoding an artificial signalpeptide, wherein the signal peptide coding sequence is operably linkedto and is in frame with the coding sequence of the antigenicpolypeptide. Thus, tropical disease vaccines of the present disclosure,in some embodiments, produce an antigenic polypeptide (e.g., a Malaria(e.g., P. falciparum, P. vivax, P. malariae and/or P. ovale), JEV, WNV,EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV antigenic polypeptide)fused to a signal peptide. In some embodiments, a signal peptide isfused to the N-terminus of the antigenic polypeptide. In someembodiments, a signal peptide is fused to the C-terminus of theantigenic polypeptide.

In some embodiments, the signal peptide fused to the antigenicpolypeptide is an artificial signal peptide. In some embodiments, anartificial signal peptide fused to the antigenic polypeptide encoded bythe RNA (e.g., mRNA) vaccine is obtained from an immunoglobulin protein,e.g., an IgE signal peptide or an IgG signal peptide. In someembodiments, a signal peptide fused to the antigenic polypeptide encodedby a RNA (e.g., mRNA) vaccine is an Ig heavy chain epsilon-1 signalpeptide (IgE HC SP) having the sequence of: MDWTWILFLVAAATRVHS; SEQ IDNO: 424. In some embodiments, a signal peptide fused to the antigenicpolypeptide encoded by the (e.g., mRNA) RNA (e.g., mRNA) vaccine is anIgGk chain V-III region HAH signal peptide (IgGk SP) having the sequenceof METPAQLLFLLLLWLPDTTG; SEQ ID NO: 423. In some embodiments, the signalpeptide is selected from: Japanese encephalitis PRM signal sequence(MLGSNSGQRVVFTILLLLVAPAYS; SEQ ID NO: 425), VSINVg protein signalsequence (MKCLLYLAFLFIGVNCA; SEQ ID NO: 426) and Japanese encephalitisJEV signal sequence (MWLVSLAIVTACAGA; SEQ ID NO: 427).

In some embodiments, the antigenic polypeptide encoded by a RNA (e.g.,mRNA) vaccine comprises an amino acid sequence identified by any one ofSEQ ID NO: 13-17, 22-29, 44-47, 52-54, 59-64, 97-117, 156-222, 469,259-291 or 402-413 fused to a signal peptide identified by any one ofSEQ ID NO: 423-427. The examples disclosed herein are not meant to belimiting and any signal peptide that is known in the art to facilitatetargeting of a protein to ER for processing and/or targeting of aprotein to the cell membrane may be used in accordance with the presentdisclosure.

A signal peptide may have a length of 15-60 amino acids. For example, asignal peptide may have a length of 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,or 60 amino acids. In some embodiments, a signal peptide has a length of20-60, 25-60, 30-60, 35-60, 40-60, 45-60, 50-60, 55-60, 15-55, 20-55,25-55, 30-55, 35-55, 40-55, 45-55, 50-55, 15-50, 20-50, 25-50, 30-50,35-50, 40-50, 45-50, 15-45, 20-45, 25-45, 30-45, 35-45, 40-45, 15-40,20-40, 25-40, 30-40, 35-40, 15-35, 20-35, 25-35, 30-35, 15-30, 20-30,25-30, 15-25, 20-25, or 15-20 amino acids.

A signal peptide is typically cleaved from the nascent polypeptide atthe cleavage junction during ER processing. The mature antigenicpolypeptide produce by a tropical disease RNA (e.g., mRNA) vaccine ofthe present disclosure typically does not comprise a signal peptide.

Chemical Modifications

Tropical disease vaccines of the present disclosure, in someembodiments, comprise at least RNA (e.g. mRNA) polynucleotide having anopen reading frame encoding at least one antigenic polypeptide thatcomprises at least one chemical modification.

The terms “chemical modification” and “chemically modified” refer tomodification with respect to adenosine (A), guanosine (G), uridine (U),thymidine (T) or cytidine (C) ribonucleosides or deoxyribnucleosides inat least one of their position, pattern, percent or population.Generally, these terms do not refer to the ribonucleotide modificationsin naturally occurring 5′-terminal mRNA cap moieties. With respect to apolypeptide, the term “modification” refers to a modification relativeto the canonical set 20 amino acids. Polypeptides, as provided herein,are also considered “modified” of they contain amino acid substitutions,insertions or a combination of substitutions and insertions.

Polynucleotides (e.g., RNA polynucleotides, such as mRNApolynucleotides), in some embodiments, comprise various (more than one)different modifications. In some embodiments, a particular region of apolynucleotide contains one, two or more (optionally different)nucleoside or nucleotide modifications. In some embodiments, a modifiedRNA polynucleotide (e.g., a modified mRNA polynucleotide), introduced toa cell or organism, exhibits reduced degradation in the cell ororganism, respectively, relative to an unmodified polynucleotide. Insome embodiments, a modified RNA polynucleotide (e.g., a modified mRNApolynucleotide), introduced into a cell or organism, may exhibit reducedimmunogenicity in the cell or organism, respectively (e.g., a reducedinnate response).

Modifications of polynucleotides include, without limitation, thosedescribed herein. Polynucleotides (e.g., RNA polynucleotides, such asmRNA polynucleotides) may comprise modifications that arenaturally-occurring, non-naturally-occurring or the polynucleotide maycomprise a combination of naturally-occurring andnon-naturally-occurring modifications. Polynucleotides may include anyuseful modification, for example, of a sugar, a nucleobase, or aninternucleoside linkage (e.g., to a linking phosphate, to aphosphodiester linkage or to the phosphodiester backbone).

Polynucleotides (e.g., RNA polynucleotides, such as mRNApolynucleotides), in some embodiments, comprise non-natural modifiednucleotides that are introduced during synthesis or post-synthesis ofthe polynucleotides to achieve desired functions or properties. Themodifications may be present on an internucleotide linkages, purine orpyrimidine bases, or sugars. The modification may be introduced withchemical synthesis or with a polymerase enzyme at the terminal of achain or anywhere else in the chain. Any of the regions of apolynucleotide may be chemically modified.

The present disclosure provides for modified nucleosides and nucleotidesof a polynucleotide (e.g., RNA polynucleotides, such as mRNApolynucleotides). A “nucleoside” refers to a compound containing a sugarmolecule (e.g., a pentose or ribose) or a derivative thereof incombination with an organic base (e.g., a purine or pyrimidine) or aderivative thereof (also referred to herein as “nucleobase”). A“nucleotide” refers to a nucleoside, including a phosphate group.Modified nucleotides may by synthesized by any useful method, such as,for example, chemically, enzymatically, or recombinantly, to include oneor more modified or non-natural nucleosides. Polynucleotides maycomprise a region or regions of linked nucleosides. Such regions mayhave variable backbone linkages. The linkages may be standardphosphodiester linkages, in which case the polynucleotides wouldcomprise regions of nucleotides.

Modified nucleotide base pairing encompasses not only the standardadenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs,but also base pairs formed between nucleotides and/or modifiednucleotides comprising non-standard or modified bases, wherein thearrangement of hydrogen bond donors and hydrogen bond acceptors permitshydrogen bonding between a non-standard base and a standard base orbetween two complementary non-standard base structures. One example ofsuch non-standard base pairing is the base pairing between the modifiednucleotide inosine and adenine, cytosine or uracil. Any combination ofbase/sugar or linker may be incorporated into polynucleotides of thepresent disclosure.

Modifications of polynucleotides (e.g., RNA polynucleotides, such asmRNA polynucleotides) that are useful in the vaccines of the presentdisclosure include, but are not limited to the following:2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine;2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonylcarbamoyladenosine; N6-glycinylcarbamoyladenosine;N6-isopentenyladenosine; N6-methyladenosine;N6-threonylcarbamoyladenosine; 1,2′-O-dimethyladenosine;1-methyladenosine; 2′-O-methyladenosine; 2′-O-ribosyladenosine(phosphate); 2-methyladenosine; 2-methylthio-N6 isopentenyladenosine;2-methylthio-N6-hydroxynorvalyl carbamoyladenosine;2′-O-methyladenosine; 2′-O-ribosyladenosine (phosphate);Isopentenyladenosine; N6-(cis-hydroxyisopentenyl)adenosine;N6,2′-O-dimethyladenosine; N6,2′-O-dimethyladenosine;N6,N6,2′-O-trimethyladenosine; N6,N6-dimethyladenosine;N6-acetyladenosine; N6-hydroxynorvalylcarbamoyladenosine;N6-methyl-N6-threonylcarbamoyladenosine; 2-methyladenosine;2-methylthio-N6-isopentenyladenosine; 7-deaza-adenosine;N1-methyl-adenosine; N6, N6 (dimethyl)adenine;N6-cis-hydroxy-isopentenyl-adenosine; α-thio-adenosine; 2(amino)adenine; 2 (aminopropyl)adenine; 2 (methylthio) N6(isopentenyl)adenine; 2-(alkyl)adenine; 2-(aminoalkyl)adenine;2-(aminopropyl)adenine; 2-(halo)adenine; 2-(halo)adenine;2-(propyl)adenine; 2′-Amino-2′-deoxy-ATP; 2′-Azido-2′-deoxy-ATP;2′-Deoxy-2′-a-aminoadenosine TP; 2′-Deoxy-2′-a-azidoadenosine TP; 6(alkyl)adenine; 6 (methyl)adenine; 6-(alkyl)adenine; 6-(methyl)adenine;7 (deaza)adenine; 8 (alkenyl)adenine; 8 (alkynyl)adenine; 8(amino)adenine; 8 (thioalkyl)adenine; 8-(alkenyl)adenine;8-(alkyl)adenine; 8-(alkynyl)adenine; 8-(amino)adenine; 8-(halo)adenine;8-(hydroxyl)adenine; 8-(thioalkyl)adenine; 8-(thiol)adenine;8-azido-adeno sine; aza adenine; deaza adenine; N6 (methyl)adenine;N6-(isopentyl)adenine; 7-deaza-8-aza-adenosine; 7-methyladenine;1-Deazaadenosine TP; 2′Fluoro-N6-Bz-deoxyadenosine TP;2′-OMe-2-Amino-ATP; 2′O-methyl-N6-Bz-deoxyadenosine TP;2′-a-Ethynyladenosine TP; 2-aminoadenine; 2-Aminoadenosine TP;2-Amino-ATP; 2′-a-Trifluoromethyladenosine TP; 2-Azidoadenosine TP;2′-b-Ethynyladenosine TP; 2-Bromoadenosine TP;2′-b-Trifluoromethyladenosine TP; 2-Chloroadenosine TP;2′-Deoxy-2′,2′-difluoroadenosine TP; 2′-Deoxy-2′-a-mercaptoadenosine TP;2′-Deoxy-2′-a-thiomethoxyadenosine TP; 2′-Deoxy-2′-b-aminoadenosine TP;2′-Deoxy-2′-b-azidoadenosine TP; 2′-Deoxy-2′-b-bromoadenosine TP;2′-Deoxy-2′-b-chloroadenosine TP; 2′-Deoxy-2′-b-fluoroadenosine TP;2′-Deoxy-2′-b-iodoadenosine TP; 2′-Deoxy-2′-b-mercaptoadenosine TP;2′-Deoxy-2′-b-thiomethoxyadenosine TP; 2-Fluoroadenosine TP;2-Iodoadenosine TP; 2-Mercaptoadenosine TP; 2-methoxy-adenine;2-methylthio-adenine; 2-Trifluoromethyladenosine TP;3-Deaza-3-bromoadenosine TP; 3-Deaza-3-chloroadenosine TP;3-Deaza-3-fluoroadenosine TP; 3-Deaza-3-iodoadenosine TP;3-Deazaadenosine TP; 4′-Azidoadenosine TP; 4′-Carbocyclic adenosine TP;4′-Ethynyladenosine TP; 5′-Homo-adenosine TP; 8-Aza-ATP;8-bromo-adenosine TP; 8-Trifluoromethyladenosine TP; 9-DeazaadenosineTP; 2-aminopurine; 7-deaza-2,6-diaminopurine;7-deaza-8-aza-2,6-diaminopurine; 7-deaza-8-aza-2-aminopurine;2,6-diaminopurine; 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine;2-thiocytidine; 3-methylcytidine; 5-formylcytidine;5-hydroxymethylcytidine; 5-methylcytidine; N4-acetylcytidine;2′-O-methylcytidine; 2′-O-methylcytidine; 5,2′-O-dimethylcytidine;5-formyl-2′-O-methylcytidine; Lysidine; N4,2′-O-dimethylcytidine;N4-acetyl-2′-O-methylcytidine; N4-methylcytidine;N4,N4-Dimethyl-2′-OMe-Cytidine TP; 4-methylcytidine; 5-aza-cytidine;Pseudo-iso-cytidine; pyrrolo-cytidine; α-thio-cytidine;2-(thio)cytosine; 2′-Amino-2′-deoxy-CTP; 2′-Azido-2′-deoxy-CTP;2′-Deoxy-2′-a-aminocytidine TP; 2′-Deoxy-2′-a-azidocytidine TP; 3(deaza) 5 (aza)cytosine; 3 (methyl)cytosine; 3-(alkyl)cytosine;3-(deaza) 5 (aza)cytosine; 3-(methyl)cytidine; 4,2′-O-dimethylcytidine;5 (halo)cytosine; 5 (methyl)cytosine; 5 (propynyl)cytosine; 5(trifluoromethyl)cytosine; 5-(alkyl)cytosine; 5-(alkynyl)cytosine;5-(halo)cytosine; 5-(propynyl)cytosine; 5-(trifluoromethyl)cytosine;5-bromo-cytidine; 5-iodo-cytidine; 5-propynyl cytosine; 6-(azo)cytosine;6-aza-cytidine; aza cytosine; deaza cytosine; N4 (acetyl)cytosine;1-methyl-1-deaza-pseudoisocytidine; 1-methyl-pseudoisocytidine;2-methoxy-5-methyl-cytidine; 2-methoxy-cytidine;2-thio-5-methyl-cytidine; 4-methoxy-1-methyl-pseudoisocytidine;4-methoxy-pseudoisocytidine; 4-thio-1-methyl-1-deaza-pseudoisocytidine;4-thio-1-methyl-pseudoisocytidine; 4-thio-pseudoisocytidine;5-aza-zebularine; 5-methyl-zebularine; pyrrolo-pseudoisocytidine;Zebularine; (E)-5-(2-Bromo-vinyl)cytidine TP; 2,2′-anhydro-cytidine TPhydrochloride; 2′Fluor-N4-Bz-cytidine TP; 2′Fluoro-N4-Acetyl-cytidineTP; 2′-O-Methyl-N4-Acetyl-cytidine TP; 2′O-methyl-N4-Bz-cytidine TP;2′-a-Ethynylcytidine TP; 2′-a-Trifluoromethylcytidine TP;2′-b-Ethynylcytidine TP; 2′-b-Trifluoromethylcytidine TP;2′-Deoxy-2′,2′-difluorocytidine TP; 2′-Deoxy-2′-a-mercaptocytidine TP;2′-Deoxy-2′-a-thiomethoxycytidine TP; 2′-Deoxy-2′-b-aminocytidine TP;2′-Deoxy-2′-b-azidocytidine TP; 2′-Deoxy-2′-b-bromocytidine TP;2′-Deoxy-2′-b-chlorocytidine TP; 2′-Deoxy-2′-b-fluorocytidine TP;2′-Deoxy-2′-b-iodocytidine TP; 2′-Deoxy-2′-b-mercaptocytidine TP;2′-Deoxy-2′-b-thiomethoxycytidine TP; 2′-O-Methyl-5-(1-propynyl)cytidineTP; 3′-Ethynylcytidine TP; 4′-Azidocytidine TP; 4′-Carbocyclic cytidineTP; 4′-Ethynylcytidine TP; 5-(1-Propynyl)ara-cytidine TP;5-(2-Chloro-phenyl)-2-thiocytidine TP; 5-(4-Amino-phenyl)-2-thiocytidineTP; 5-Aminoallyl-CTP; 5-Cyanocytidine TP; 5-Ethynylara-cytidine TP;5-Ethynylcytidine TP; 5′-Homo-cytidine TP; 5-Methoxycytidine TP;5-Trifluoromethyl-Cytidine TP; N4-Amino-cytidine TP; N4-Benzoyl-cytidineTP; Pseudoisocytidine; 7-methylguanosine; N2,2′-O-dimethylguanosine;N2-methylguanosine; Wyosine; 1,2′-O-dimethylguanosine;1-methylguanosine; 2′-O-methylguanosine; 2′-O-ribosylguanosine(phosphate); 2′-O-methylguanosine; 2′-O-ribosylguanosine (phosphate);7-aminomethyl-7-deazaguanosine; 7-cyano-7-deazaguanosine; Archaeosine;Methylwyo sine; N2,7-dimethylguanosine; N2,N2,2′-O-trimethylguanosine;N2,N2,7-trimethylguanosine; N2,N2-dimethylguanosine;N2,7,2′-O-trimethylguanosine; 6-thio-guanosine; 7-deaza-guanosine;8-oxo-guanosine; N1-methyl-guanosine; α-thio-guanosine; 2(propyl)guanine; 2-(alkyl)guanine; 2′-Amino-2′-deoxy-GTP;2′-Azido-2′-deoxy-GTP; 2′-Deoxy-2′-a-aminoguanosine TP;2′-Deoxy-2′-a-azidoguanosine TP; 6 (methyl)guanine; 6-(alkyl)guanine;6-(methyl)guanine; 6-methyl-guanosine; 7 (alkyl)guanine; 7(deaza)guanine; 7 (methyl)guanine; 7-(alkyl)guanine; 7-(deaza)guanine;7-(methyl)guanine; 8 (alkyl)guanine; 8 (alkynyl)guanine; 8(halo)guanine; 8 (thioalkyl)guanine; 8-(alkenyl)guanine;8-(alkyl)guanine; 8-(alkynyl)guanine; 8-(amino)guanine; 8-(halo)guanine;8-(hydroxyl)guanine; 8-(thioalkyl)guanine; 8-(thiol)guanine; azaguanine; deaza guanine; N (methyl)guanine; N-(methyl)guanine;1-methyl-6-thio-guanosine; 6-methoxy-guanosine;6-thio-7-deaza-8-aza-guanosine; 6-thio-7-deaza-guanosine;6-thio-7-methyl-guanosine; 7-deaza-8-aza-guanosine;7-methyl-8-oxo-guanosine; N2,N2-dimethyl-6-thio-guanosine;N2-methyl-6-thio-guanosine; 1-Me-GTP; 2′Fluoro-N2-isobutyl-guanosine TP;2′O-methyl-N2-isobutyl-guanosine TP; 2′-a-Ethynylguanosine TP;2′-a-Trifluoromethylguanosine TP; 2′-b-Ethynylguanosine TP;2′-b-Trifluoromethylguanosine TP; 2′-Deoxy-2′,2′-difluoroguanosine TP;2′-Deoxy-2′-a-mercaptoguanosine TP; 2′-Deoxy-2′-a-thiomethoxyguanosineTP; 2′-Deoxy-2′-b-aminoguanosine TP; 2′-Deoxy-2′-b-azidoguanosine TP;2′-Deoxy-2′-b-bromoguanosine TP; 2′-Deoxy-2′-b-chloroguanosine TP;2′-Deoxy-2′-b-fluoroguanosine TP; 2′-Deoxy-2′-b-iodoguanosine TP;2′-Deoxy-2′-b-mercaptoguanosine TP; 2′-Deoxy-2′-b-thiomethoxyguanosineTP; 4′-Azidoguanosine TP; 4′-Carbocyclic guanosine TP;4′-Ethynylguanosine TP; 5′-Homo-guanosine TP; 8-bromo-guanosine TP;9-Deazaguanosine TP; N2-isobutyl-guanosine TP; 1-methylinosine; Inosine;1,2′-O-dimethylinosine; 2′-O-methylinosine; 7-methylinosine;2′-O-methylinosine; Epoxyqueuosine; galactosyl-queuosine;Mannosylqueuosine; Queuosine; allyamino-thymidine; aza thymidine; deazathymidine; deoxy-thymidine; 2′-O-methyluridine; 2-thiouridine;3-methyluridine; 5-carboxymethyluridine; 5-hydroxyuridine;5-methyluridine; 5-taurinomethyl-2-thiouridine; 5-taurinomethyluridine;Dihydrouridine; Pseudouridine; (3-(3-amino-3-carboxypropyl)uridine;1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine;1-methylpseduouridine; 1-methyl-pseudouridine; 2′-O-methyluridine;2′-O-methylpseudouridine; 2′-O-methyluridine; 2-thio-2′-O-methyluridine;3-(3-amino-3-carboxypropyl)uridine; 3,2′-O-dimethyluridine;3-Methyl-pseudo-Uridine TP; 4-thiouridine;5-(carboxyhydroxymethyl)uridine; 5-(carboxyhydroxymethyl)uridine methylester; 5,2′-O-dimethyluridine; 5,6-dihydro-uridine;5-aminomethyl-2-thiouridine; 5-carbamoylmethyl-2′-O-methyluridine;5-carbamoylmethyluridine; 5-carboxyhydroxymethyluridine;5-carboxyhydroxymethyluridine methyl ester;5-carboxymethylaminomethyl-2′-O-methyluridine;5-carboxymethylaminomethyl-2-thiouridine;5-carboxymethylaminomethyl-2-thiouridine;5-carboxymethylaminomethyluridine; 5-carboxymethylaminomethyluridine;5-Carbamoylmethyluridine TP; 5-methoxycarbonylmethyl-2′-O-methyluridine;5-methoxycarbonylmethyl-2-thiouridine; 5-methoxycarbonylmethyluridine;5-methoxyuridine; 5-methyl-2-thiouridine;5-methylaminomethyl-2-selenouridine; 5-methylaminomethyl-2-thiouridine;5-methylaminomethyluridine; 5-Methyldihydrouridine; 5-Oxyaceticacid-Uridine TP; 5-Oxyacetic acid-methyl ester-Uridine TP;N1-methyl-pseudo-uridine; uridine 5-oxyacetic acid; uridine 5-oxyaceticacid methyl ester; 3-(3-Amino-3-carboxypropyl)-Uridine TP;5-(iso-Pentenylaminomethyl)-2-thiouridine TP;5-(iso-Pentenylaminomethyl)-2′-O-methyluridine TP;5-(iso-Pentenylaminomethyl)uridine TP; 5-propynyl uracil;α-thio-uridine; 1(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil; 1(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1(aminoalkylaminocarbonylethylenyl)-4 (thio)pseudouracil; 1(aminoalkylaminocarbonylethylenyl)-pseudouracil; 1(aminocarbonylethylenyl)-2(thio)-pseudouracil; 1(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1(aminocarbonylethylenyl)-4 (thio)pseudouracil; 1(aminocarbonylethylenyl)-pseudouracil; 1 substituted2(thio)-pseudouracil; 1 substituted 2,4-(dithio)pseudouracil; 1substituted 4 (thio)pseudouracil; 1 substituted pseudouracil;1-(aminoalkylamino-carbonylethylenyl)-2-(thio)-pseudouracil;1-Methyl-3-(3-amino-3-carboxypropyl) pseudouridine TP;1-Methyl-3-(3-amino-3-carboxypropyl)pseudo-UTP; 1-Methyl-pseudo-UTP; 2(thio)pseudouracil; 2′ deoxy uridine; 2′ fluorouridine; 2-(thio)uracil;2,4-(dithio)psuedouracil; 2′ methyl, 2′amino, 2′azido,2′fluro-guanosine; 2′-Amino-2′-deoxy-UTP; 2′-Azido-2′-deoxy-UTP;2′-Azido-deoxyuridine TP; 2′-O-methylpseudouridine; 2′ deoxy uridine; 2′fluorouridine; 2′-Deoxy-2′-a-aminouridine TP; 2′-Deoxy-2′-a-azidouridineTP; 2-methylpseudouridine; 3 (3 amino-3 carboxypropyl)uracil; 4(thio)pseudouracil; 4-(thio)pseudouracil; 4-(thio)uracil; 4-thiouracil;5 (1,3-diazole-1-alkyl)uracil; 5 (2-aminopropyl)uracil; 5(aminoalkyl)uracil; 5 (dimethylaminoalkyl)uracil; 5(guanidiniumalkyl)uracil; 5 (methoxycarbonylmethyl)-2-(thio)uracil; 5(methoxycarbonyl-methyl)uracil; 5 (methyl) 2 (thio)uracil; 5 (methyl)2,4 (dithio)uracil; 5 (methyl) 4 (thio)uracil; 5 (methylaminomethyl)-2(thio)uracil; 5 (methylaminomethyl)-2,4 (dithio)uracil; 5(methylaminomethyl)-4 (thio)uracil; 5 (propynyl)uracil; 5(trifluoromethyl)uracil; 5-(2-aminopropyl)uracil;5-(alkyl)-2-(thio)pseudouracil; 5-(alkyl)-2,4 (dithio)pseudouracil;5-(alkyl)-4 (thio)pseudouracil; 5-(alkyl)pseudouracil; 5-(alkyl)uracil;5-(alkynyl)uracil; 5-(allylamino)uracil; 5-(cyanoalkyl)uracil;5-(dialkylaminoalkyl)uracil; 5-(dimethylaminoalkyl)uracil;5-(guanidiniumalkyl)uracil; 5-(halo)uracil;5-(1,3-diazole-1-alkyl)uracil; 5-(methoxy)uracil;5-(methoxycarbonylmethyl)-2-(thio)uracil;5-(methoxycarbonyl-methyl)uracil; 5-(methyl) 2(thio)uracil; 5-(methyl)2,4 (dithio)uracil; 5-(methyl) 4 (thio)uracil;5-(methyl)-2-(thio)pseudouracil; 5-(methyl)-2,4 (dithio)pseudouracil;5-(methyl)-4 (thio)pseudouracil; 5-(methyl)pseudouracil;5-(methylaminomethyl)-2 (thio)uracil;5-(methylaminomethyl)-2,4(dithio)uracil;5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil;5-(trifluoromethyl)uracil; 5-aminoallyl-uridine; 5-bromo-uridine;5-iodo-uridine; 5-uracil; 6 (azo)uracil; 6-(azo)uracil; 6-aza-uridine;allyamino-uracil; aza uracil; deaza uracil; N3 (methyl)uracil;Pseudo-UTP-1-2-ethanoic acid; Pseudouracil; 4-Thio-pseudo-UTP;1-carboxymethyl-pseudouridine; 1-methyl-1-deaza-pseudouridine;1-propynyl-uridine; 1-taurinomethyl-1-methyl-uridine;1-taurinomethyl-4-thio-uridine; 1-taurinomethyl-pseudouridine;2-methoxy-4-thio-pseudouridine; 2-thio-1-methyl-1-deaza-pseudouridine;2-thio-1-methyl-pseudouridine; 2-thio-5-aza-uridine;2-thio-dihydropseudouridine; 2-thio-dihydrouridine;2-thio-pseudouridine; 4-methoxy-2-thio-pseudouridine;4-methoxy-pseudouridine; 4-thio-1-methyl-pseudouridine;4-thio-pseudouridine; 5-aza-uridine; Dihydropseudouridine;(±)1-(2-Hydroxypropyl)pseudouridine TP;(2R)-1-(2-Hydroxypropyl)pseudouridine TP;(2S)-1-(2-Hydroxypropyl)pseudouridine TP;(E)-5-(2-Bromo-vinyl)ara-uridine TP; (E)-5-(2-Bromo-vinyl)uridine TP;(Z)-5-(2-Bromo-vinyl)ara-uridine TP; (Z)-5-(2-Bromo-vinyl)uridine TP;1-(2,2,2-Trifluoroethyl)-pseudo-UTP;1-(2,2,3,3,3-Pentafluoropropyl)pseudouridine TP;1-(2,2-Diethoxyethyl)pseudouridine TP;1-(2,4,6-Trimethylbenzyl)pseudouridine TP;1-(2,4,6-Trimethyl-benzyl)pseudo-UTP;1-(2,4,6-Trimethyl-phenyl)pseudo-UTP;1-(2-Amino-2-carboxyethyl)pseudo-UTP; 1-(2-Amino-ethyl)pseudo-UTP;1-(2-Hydroxyethyl)pseudouridine TP; 1-(2-Methoxyethyl)pseudouridine TP;1-(3,4-Bis-trifluoromethoxybenzyl)pseudouridine TP;1-(3,4-Dimethoxybenzyl)pseudouridine TP;1-(3-Amino-3-carboxypropyl)pseudo-UTP; 1-(3-Amino-propyl)pseudo-UTP;1-(3-Cyclopropyl-prop-2-ynyl)pseudouridine TP;1-(4-Amino-4-carboxybutyl)pseudo-UTP; 1-(4-Amino-benzyl)pseudo-UTP;1-(4-Amino-butyl)pseudo-UTP; 1-(4-Amino-phenyl)pseudo-UTP;1-(4-Azidobenzyl)pseudouridine TP; 1-(4-Bromobenzyl)pseudouridine TP;1-(4-Chlorobenzyl)pseudouridine TP; 1-(4-Fluorobenzyl)pseudouridine TP;1-(4-Iodobenzyl)pseudouridine TP;1-(4-Methanesulfonylbenzyl)pseudouridine TP;1-(4-Methoxybenzyl)pseudouridine TP; 1-(4-Methoxy-benzyl)pseudo-UTP;1-(4-Methoxy-phenyl)pseudo-UTP; 1-(4-Methylbenzyl)pseudouridine TP;1-(4-Methyl-benzyl)pseudo-UTP; 1-(4-Nitrobenzyl)pseudouridine TP;1-(4-Nitro-benzyl)pseudo-UTP; 1(4-Nitro-phenyl)pseudo-UTP;1-(4-Thiomethoxybenzyl)pseudouridine TP;1-(4-Trifluoromethoxybenzyl)pseudouridine TP;1-(4-Trifluoromethylbenzyl)pseudouridine TP;1-(5-Amino-pentyl)pseudo-UTP; 1-(6-Amino-hexyl)pseudo-UTP;1,6-Dimethyl-pseudo-UTP;1-[3-(2-{2-[2-(2-Aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]pseudouridineTP; 1-{3-[2-(2-Aminoethoxy)-ethoxy]-propionyl}pseudouridine TP;1-Acetylpseudouridine TP; 1-Alkyl-6-(1-propynyl)-pseudo-UTP;1-Alkyl-6-(2-propynyl)-pseudo-UTP; 1-Alkyl-6-allyl-pseudo-UTP;1-Alkyl-6-ethynyl-pseudo-UTP; 1-Alkyl-6-homoallyl-pseudo-UTP;1-Alkyl-6-vinyl-pseudo-UTP; 1-Allylpseudouridine TP;1-Aminomethyl-pseudo-UTP; 1-Benzoylpseudouridine TP;1-Benzyloxymethylpseudouridine TP; 1-Benzyl-pseudo-UTP;1-Biotinyl-PEG2-pseudouridine TP; 1-Biotinylpseudouridine TP;1-Butyl-pseudo-UTP; 1-Cyanomethylpseudouridine TP;1-Cyclobutylmethyl-pseudo-UTP; 1-Cyclobutyl-pseudo-UTP;1-Cycloheptylmethyl-pseudo-UTP; 1-Cycloheptyl-pseudo-UTP;1-Cyclohexylmethyl-pseudo-UTP; 1-Cyclohexyl-pseudo-UTP;1-Cyclooctylmethyl-pseudo-UTP; 1-Cyclooctyl-pseudo-UTP;1-Cyclopentylmethyl-pseudo-UTP; 1-Cyclopentyl-pseudo-UTP;1-Cyclopropylmethyl-pseudo-UTP; 1-Cyclopropyl-pseudo-UTP;1-Ethyl-pseudo-UTP; 1-Hexyl-pseudo-UTP; 1-Homoallylpseudouridine TP;1-Hydroxymethylpseudouridine TP; 1-iso-propyl-pseudo-UTP;1-Me-2-thio-pseudo-UTP; 1-Me-4-thio-pseudo-UTP;1-Me-alpha-thio-pseudo-UTP; 1-Methanesulfonylmethylpseudouridine TP;1-Methoxymethylpseudouridine TP;1-Methyl-6-(2,2,2-Trifluoroethyl)pseudo-UTP;1-Methyl-6-(4-morpholino)-pseudo-UTP;1-Methyl-6-(4-thiomorpholino)-pseudo-UTP; 1-Methyl-6-(substitutedphenyl)pseudo-UTP; 1-Methyl-6-amino-pseudo-UTP;1-Methyl-6-azido-pseudo-UTP; 1-Methyl-6-bromo-pseudo-UTP;1-Methyl-6-butyl-pseudo-UTP; 1-Methyl-6-chloro-pseudo-UTP;1-Methyl-6-cyano-pseudo-UTP; 1-Methyl-6-dimethylamino-pseudo-UTP;1-Methyl-6-ethoxy-pseudo-UTP; 1-Methyl-6-ethylcarboxylate-pseudo-UTP;1-Methyl-6-ethyl-pseudo-UTP; 1-Methyl-6-fluoro-pseudo-UTP;1-Methyl-6-formyl-pseudo-UTP; 1-Methyl-6-hydroxyamino-pseudo-UTP;1-Methyl-6-hydroxy-pseudo-UTP; 1-Methyl-6-iodo-pseudo-UTP;1-Methyl-6-iso-propyl-pseudo-UTP; 1-Methyl-6-methoxy-pseudo-UTP;1-Methyl-6-methylamino-pseudo-UTP; 1-Methyl-6-phenyl-pseudo-UTP;1-Methyl-6-propyl-pseudo-UTP; 1-Methyl-6-tert-butyl-pseudo-UTP;1-Methyl-6-trifluoromethoxy-pseudo-UTP;1-Methyl-6-trifluoromethyl-pseudo-UTP; 1-MorpholinomethylpseudouridineTP; 1-Pentyl-pseudo-UTP; 1-Phenyl-pseudo-UTP; 1-PivaloylpseudouridineTP; 1-Propargylpseudouridine TP; 1-Propyl-pseudo-UTP;1-propynyl-pseudouridine; 1-p-tolyl-pseudo-UTP; 1-tert-Butyl-pseudo-UTP;1-Thiomethoxymethylpseudouridine TP; 1-ThiomorpholinomethylpseudouridineTP; 1-Trifluoroacetylpseudouridine TP; 1-Trifluoromethyl-pseudo-UTP;1-Vinylpseudouridine TP; 2,2′-anhydro-uridine TP; 2′-bromo-deoxyuridineTP; 2′-F-5-Methyl-2′-deoxy-UTP; 2′-OMe-5-Me-UTP; 2′-OMe-pseudo-UTP;2′-a-Ethynyluridine TP; 2′-a-Trifluoromethyluridine TP;2′-b-Ethynyluridine TP; 2′-b-Trifluoromethyluridine TP;2′-Deoxy-2′,2′-difluorouridine TP; 2′-Deoxy-2′-a-mercaptouridine TP;2′-Deoxy-2′-a-thiomethoxyuridine TP; 2′-Deoxy-2′-b-aminouridine TP;2′-Deoxy-2′-b-azidouridine TP; 2′-Deoxy-2′-b-bromouridine TP;2′-Deoxy-2′-b-chlorouridine TP; 2′-Deoxy-2′-b-fluorouridine TP;2′-Deoxy-2′-b-iodouridine TP; 2′-Deoxy-2′-b-mercaptouridine TP;2′-Deoxy-2′-b-thiomethoxyuridine TP; 2-methoxy-4-thio-uridine;2-methoxyuridine; 2′-O-Methyl-5-(1-propynyl)uridine TP;3-Alkyl-pseudo-UTP; 4′-Azidouridine TP; 4′-Carbocyclic uridine TP;4′-Ethynyluridine TP; 5-(1-Propynyl)ara-uridine TP; 5-(2-Furanyl)uridineTP; 5-Cyanouridine TP; 5-Dimethylaminouridine TP; 5′-Homo-uridine TP;5-iodo-2′-fluoro-deoxyuridine TP; 5-Phenylethynyluridine TP;5-Trideuteromethyl-6-deuterouridine TP; 5-Trifluoromethyl-Uridine TP;5-Vinylarauridine TP; 6-(2,2,2-Trifluoroethyl)-pseudo-UTP;6-(4-Morpholino)-pseudo-UTP; 6-(4-Thiomorpholino)-pseudo-UTP;6-(Substituted-Phenyl)-pseudo-UTP; 6-Amino-pseudo-UTP;6-Azido-pseudo-UTP; 6-Bromo-pseudo-UTP; 6-Butyl-pseudo-UTP;6-Chloro-pseudo-UTP; 6-Cyano-pseudo-UTP; 6-Dimethylamino-pseudo-UTP;6-Ethoxy-pseudo-UTP; 6-Ethylcarboxylate-pseudo-UTP; 6-Ethyl-pseudo-UTP;6-Fluoro-pseudo-UTP; 6-Formyl-pseudo-UTP; 6-Hydroxyamino-pseudo-UTP;6-Hydroxy-pseudo-UTP; 6-Iodo-pseudo-UTP; 6-iso-Propyl-pseudo-UTP;6-Methoxy-pseudo-UTP; 6-Methylamino-pseudo-UTP; 6-Methyl-pseudo-UTP;6-Phenyl-pseudo-UTP; 6-Phenyl-pseudo-UTP; 6-Propyl-pseudo-UTP;6-tert-Butyl-pseudo-UTP; 6-Trifluoromethoxy-pseudo-UTP;6-Trifluoromethyl-pseudo-UTP; Alpha-thio-pseudo-UTP; Pseudouridine1-(4-methylbenzenesulfonic acid) TP; Pseudouridine 1-(4-methylbenzoicacid) TP; Pseudouridine TP 1-[3-(2-ethoxy)]propionic acid; PseudouridineTP 1-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy}]propionic acid;Pseudouridine TP1-[3-{2-(2-[2-{2(2-ethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}]propionicacid; Pseudouridine TP 1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionicacid; Pseudouridine TP 1-[3-{2-(2-ethoxy)-ethoxy}]propionic acid;Pseudouridine TP 1-methylphosphonic acid; Pseudouridine TP1-methylphosphonic acid diethyl ester; Pseudo-UTP-N1-3-propionic acid;Pseudo-UTP-N1-4-butanoic acid; Pseudo-UTP-N1-5-pentanoic acid;Pseudo-UTP-N1-6-hexanoic acid; Pseudo-UTP-N1-7-heptanoic acid;Pseudo-UTP-N1-methyl-p-benzoic acid; Pseudo-UTP-N1-p-benzoic acid;Wybutosine; Hydroxywybutosine; Isowyosine; Peroxywybutosine;undermodified hydroxywybutosine; 4-demethylwyosine; 2,6-(diamino)purine;1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl:1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;1,3,5-(triaza)-2,6-(dioxa)-naphthalene; 2 (amino)purine;2,4,5-(trimethyl)phenyl; 2′ methyl, 2′amino, 2′azido, 2′fluro-cytidine;2′ methyl, 2′ amino, 2′ azido, 2′fluro-adenine; 2′methyl, 2′ amino, 2′azido, 2′fluro-uridine; 2′-amino-2′-deoxyribose;2-amino-6-Chloro-purine; 2-aza-inosinyl; 2′-azido-2′-deoxyribose;2′fluoro-2′-deoxyribose; 2′-fluoro-modified bases; 2′-O-methyl-ribose;2-oxo-7-aminopyridopyrimidin-3-yl; 2-oxo-pyridopyrimidine-3-yl;2-pyridinone; 3 nitropyrrole; 3-(methyl)-7-(propynyl)isocarbostyrilyl;3-(methyl)isocarbostyrilyl; 4-(fluoro)-6-(methyl)benzimidazole;4-(methyl)benzimidazole; 4-(methyl)indolyl; 4,6-(dimethyl)indolyl; 5nitroindole; 5 substituted pyrimidines; 5-(methyl)isocarbostyrilyl;5-nitroindole; 6-(aza)pyrimidine; 6-(azo)thymine;6-(methyl)-7-(aza)indolyl; 6-chloro-purine;6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl;7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(aza)indolyl;7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazinl-yl;7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl;7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(propynyl)isocarbostyrilyl; 7-(propynyl)isocarbostyrilyl,propynyl-7-(aza)indolyl; 7-deaza-inosinyl; 7-substituted1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-substituted1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 9-(methyl)-imidizopyridinyl;Aminoindolyl; Anthracenyl;bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;Difluorotolyl; Hypoxanthine; Imidizopyridinyl; Inosinyl;Isocarbostyrilyl; Isoguanisine; N2-substituted purines;N6-methyl-2-amino-purine; N6-substituted purines; N-alkylatedderivative; Napthalenyl; Nitrobenzimidazolyl; Nitroimidazolyl;Nitroindazolyl; Nitropyrazolyl; Nubularine; 06-substituted purines;O-alkylated derivative;ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Oxoformycin TP;para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Pentacenyl;Phenanthracenyl; Phenyl; propynyl-7-(aza)indolyl; Pyrenyl;pyridopyrimidin-3-yl; pyridopyrimidin-3-yl,2-oxo-7-aminopyridopyrimidin-3-yl; pyrrolo-pyrimidin-2-on-3-yl;Pyrrolopyrimidinyl; Pyrrolopyrizinyl; Stilbenzyl; substituted1,2,4-triazoles; Tetracenyl; Tubercidine; Xanthine; Xanthosine-5′-TP;2-thio-zebularine; 5-aza-2-thio-zebularine; 7-deaza-2-amino-purine;pyridin-4-one ribonucleoside; 2-Amino-riboside-TP; Formycin A TP;Formycin B TP; Pyrrolosine TP; 2′-OH-ara-adenosine TP;2′-OH-ara-cytidine TP; 2′-OH-ara-uridine TP; 2′-OH-ara-guanosine TP;5-(2-carbomethoxyvinyl)uridine TP; andN6-(19-Amino-pentaoxanonadecyl)adenosine TP.

In some embodiments, polynucleotides (e.g., RNA polynucleotides, such asmRNA polynucleotides) include a combination of at least two (e.g., 2, 3,4 or more) of the aforementioned modified nucleobases.

In some embodiments, modified nucleobases in polynucleotides (e.g., RNApolynucleotides, such as mRNA polynucleotides) are selected from thegroup consisting of pseudouridine (ψ), N1-methylpseudouridine (m¹ψ),N1-ethylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine,2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine,2-thio-5-aza-uridine, 2-thio-dihydropseudouridine,2-thio-dihydrouridine, 2-thio-pseudouridine,4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine,4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine,dihydropseudouridine, 5-methoxyuridine and 2′-O-methyl uridine. In someembodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNApolynucleotides) include a combination of at least two (e.g., 2, 3, 4 ormore) of the aforementioned modified nucleobases.

In some embodiments, modified nucleobases in polynucleotides (e.g., RNApolynucleotides, such as mRNA polynucleotides) are selected from thegroup consisting of 1-methyl-pseudouridine (m¹ψ), 5-methoxy-uridine(mo⁵U), 5-methyl-cytidine (m⁵C), pseudouridine (ψ), α-thio-guanosine andα-thio-adenosine. In some embodiments, polynucleotides (e.g., RNApolynucleotides, such as mRNA polynucleotides) include a combination ofat least two (e.g., 2, 3, 4 or more) of the aforementioned modifiednucleobases.

In some embodiments, polynucleotides (e.g., RNA polynucleotides, such asmRNA polynucleotides) comprise pseudouridine (ψ) and 5-methyl-cytidine(m⁵C). In some embodiments, polynucleotides (e.g., RNA polynucleotides,such as mRNA polynucleotides) comprise 1-methyl-pseudouridine (m¹ψ). Insome embodiments, polynucleotides (e.g., RNA polynucleotides, such asmRNA polynucleotides) comprise 1-methyl-pseudouridine (m¹ψ) and5-methyl-cytidine (m⁵C). In some embodiments, polynucleotides (e.g., RNApolynucleotides, such as mRNA polynucleotides) comprise 2-thiouridine(s²U). In some embodiments, polynucleotides (e.g., RNA polynucleotides,such as mRNA polynucleotides) comprise 2-thiouridine and5-methyl-cytidine (m⁵C). In some embodiments, polynucleotides (e.g., RNApolynucleotides, such as mRNA polynucleotides) comprise methoxy-uridine(mo⁵U). In some embodiments, polynucleotides (e.g., RNA polynucleotides,such as mRNA polynucleotides) comprise 5-methoxy-uridine (mo⁵U) and5-methyl-cytidine (m⁵C). In some embodiments, polynucleotides (e.g., RNApolynucleotides, such as mRNA polynucleotides) comprise 2′-O-methyluridine. In some embodiments polynucleotides (e.g., RNA polynucleotides,such as mRNA polynucleotides) comprise 2′-O-methyl uridine and5-methyl-cytidine (m⁵C). In some embodiments, polynucleotides (e.g., RNApolynucleotides, such as mRNA polynucleotides) compriseN6-methyl-adenosine (m⁶A). In some embodiments, polynucleotides (e.g.,RNA polynucleotides, such as mRNA polynucleotides) compriseN6-methyl-adenosine (m⁶A) and 5-methyl-cytidine (m⁵C).

In some embodiments, polynucleotides (e.g., RNA polynucleotides, such asmRNA polynucleotides) are uniformly modified (e.g., fully modified,modified throughout the entire sequence) for a particular modification.For example, a polynucleotide can be uniformly modified with5-methyl-cytidine (m⁵C), meaning that all cytosine residues in the mRNAsequence are replaced with 5-methyl-cytidine (m⁵C). Similarly, apolynucleotide can be uniformly modified for any type of nucleosideresidue present in the sequence by replacement with a modified residuesuch as those set forth above.

Exemplary nucleobases and nucleosides having a modified cytosine includeN4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine(e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C),1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), and2-thio-5-methyl-cytidine.

In some embodiments, a modified nucleobase is a modified uridine. Insome embodiments, a modified nucleobase is a modified cytosine.Nucleosides having a modified uridine include 5-cyano uridine, and4′-thio uridine.

In some embodiments, a modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having a modified adenine include7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), andN6-methyl-adenosine (m6A).

In some embodiments, a modified nucleobase is a modified guanine.Exemplary nucleobases and nucleosides having a modified guanine includeinosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine(mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0),7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G),1-methyl-guanosine (m1G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine.

The polynucleotides of the present disclosure may be partially or fullymodified along the entire length of the molecule. For example, one ormore or all or a given type of nucleotide (e.g., purine or pyrimidine,or any one or more or all of A, G, U, C) may be uniformly modified in apolynucleotide of the invention, or in a given predetermined sequenceregion thereof (e.g., in the mRNA including or excluding the polyAtail). In some embodiments, all nucleotides X in a polynucleotide of thepresent disclosure (or in a given sequence region thereof) are modifiednucleotides, wherein X may any one of nucleotides A, G, U, C, or any oneof the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C orA+G+C.

The polynucleotide may contain from about 1% to about 100% modifiednucleotides (either in relation to overall nucleotide content, or inrelation to one or more types of nucleotide, i.e., any one or more of A,G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1%to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%,from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10%to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%,from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%,from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%,from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%,from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%,from 90% to 100%, and from 95% to 100%). Any remaining percentage isaccounted for by the presence of unmodified A, G, U, or C.

The polynucleotides may contain at a minimum 1% and at maximum 100%modified nucleotides, or any intervening percentage, such as at least 5%modified nucleotides, at least 10% modified nucleotides, at least 25%modified nucleotides, at least 50% modified nucleotides, at least 80%modified nucleotides, or at least 90% modified nucleotides. For example,the polynucleotides may contain a modified pyrimidine such as a modifieduracil or cytosine. In some embodiments, at least 5%, at least 10%, atleast 25%, at least 50%, at least 80%, at least 90% or 100% of theuracil in the polynucleotide is replaced with a modified uracil (e.g., a5-substituted uracil). The modified uracil can be replaced by a compoundhaving a single unique structure, or can be replaced by a plurality ofcompounds having different structures (e.g., 2, 3, 4 or more uniquestructures). In some embodiments, at least 5%, at least 10%, at least25%, at least 50%, at least 80%, at least 90% or 100% of the cytosine inthe polynucleotide is replaced with a modified cytosine (e.g., a5-substituted cytosine). The modified cytosine can be replaced by acompound having a single unique structure, or can be replaced by aplurality of compounds having different structures (e.g., 2, 3, 4 ormore unique structures).

Thus, in some embodiments, the RNA (e.g., mRNA) vaccines comprise a5′UTR element, an optionally codon optimized open reading frame, and a3′UTR element, a poly(A) sequence and/or a polyadenylation signalwherein the RNA is not chemically modified.

In some embodiments, the modified nucleobase is a modified uracil.Exemplary nucleobases and nucleosides having a modified uracil includepseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine,6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s²U),4-thio-uridine (s⁴U), 4-thio-pseudouridine, 2-thio-pseudouridine,5-hydroxy-uridine (ho⁵U), 5-aminoallyl-uridine, 5-halo-uridine (e.g.,5-iodo-uridineor 5-bromo-uridine), 3-methyl-uridine (m³U),5-methoxy-uridine (mo⁵U), uridine 5-oxyacetic acid (cmo⁵U), uridine5-oxyacetic acid methyl ester (mcmo⁵U), 5-carboxymethyl-uridine (cm⁵U),1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm⁵U),5-carboxyhydroxymethyl-uridine methyl ester (mchm⁵U),5-methoxycarbonylmethyl-uridine (mcm⁵U),5-methoxycarbonylmethyl-2-thio-uridine (mcm⁵s²U),5-aminomethyl-2-thio-uridine (nm⁵s²U), 5-methylaminomethyl-uridine(mnm⁵U), 5-methylaminomethyl-2-thio-uridine (mnm⁵s²U),5-methylaminomethyl-2-seleno-uridine (mnm⁵se²U),5-carbamoylmethyl-uridine (ncm⁵U), 5-carboxymethylaminomethyl-uridine(cmnm⁵U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm⁵s²U),5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine(™⁵U), 1-taurinomethyl-pseudouridine,5-taurinomethyl-2-thio-uridine(τm⁵s²U),1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m⁵U, i.e.,having the nucleobase deoxythymine), 1-methyl-pseudouridine (m¹ψ),5-methyl-2-thio-uridine (m⁵s²U), 1-methyl-4-thio-pseudouridine(m¹s⁴ψ),4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ψ),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D),dihydropseudouridine, 5,6-dihydrouridine, 5-methyldihydrouridine (m⁵D),2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine,2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine,4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine,3-(3-amino-3-carboxypropyl)uridine (acp³U),1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ψ),5-(isopentenylaminomethyl)uridine (inm⁵U),5-(isopentenylaminomethyl)-2-thio-uridine (inm⁵s²U), α-thio-uridine,2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m⁵Um),2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O-methyl-uridine (s²Um),5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm⁵Um),5-carbamoylmethyl-2′-O-methyl-uridine (ncm⁵Um),5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm⁵Um),3,2′-O-dimethyl-uridine (m³Um), and5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm⁵Um), 1-thio-uridine,deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-0H-ara-uridine,5-(2-carbomethoxyvinyl) uridine, and 5-[3-(1-E-propenylamino)]uridine.

In some embodiments, the modified nucleobase is a modified cytosine.Exemplary nucleobases and nucleosides having a modified cytosine include5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine(m³C), N4-acetyl-cytidine (ac⁴C), 5-formylcytidine (f⁵C),N4-methyl-cytidine (m⁴C), 5-methyl-cytidine (m⁵C), 5-halo-cytidine(e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm⁵C),1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine (s²C), 2-thio-5-methyl-cytidine,4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,lysidine (k2C), α-thio-cytidine, 2′-O-methyl-cytidine (Cm),5,2′-O-dimethyl-cytidine (m⁵Cm), N4-acetyl-2′-O-methyl-cytidine (ac⁴Cm),N4,2′-O-dimethyl-cytidine (m⁴Cm), 5-formyl-2′-O-methyl-cytidine (f⁵Cm),N4,N4,2′-O-trimethyl-cytidine (m⁴ ₂Cm), 1-thio-cytidine,2′-F-ara-cytidine, 2′-F-cytidine, and 2′-OH-ara-cytidine.

In some embodiments, the modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having a modified adenine include2-amino-purine, 2, 6-diaminopurine, 2-amino-6-halo-purine (e.g.,2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine),2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine,7-deaza-8-aza-adenine, 7-deaza-2-amino-purine,7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m¹A),2-methyl-adenine (m²A), N6-methyl-adenosine (m⁶A),2-methylthio-N6-methyl-adenosine (ms² m⁶A), N6-isopentenyl-adenosine(i⁶A), 2-methylthio-N6-isopentenyl-adenosine (ms²i⁶A),N6-(cis-hydroxyisopentenyl)adenosine (io⁶A),2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms²io⁶A),N6-glycinylcarbamoyl-adenosine (g⁶A), N6-threonylcarbamoyl-adenosine(t⁶A), N6-methyl-N6-threonylcarbamoyl-adenosine (m⁶t⁶A),2-methylthio-N6-threonylcarbamoyl-adenosine (ms²g⁶A),N6,N6-dimethyl-adenosine (m⁶ ₂A), N6-hydroxynorvalylcarbamoyl-adenosine(hn⁶A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms²hn⁶A),N6-acetyl-adenosine (ac⁶A), 7-methyl-adenine, 2-methylthio-adenine,2-methoxy-adenine, α-thio-adenosine, 2′-O-methyl-adenosine (Am),N6,2′-O-dimethyl-adenosine (m⁶Am), N6,N6,2′-O-trimethyl-adenosine (m⁶₂Am), 1,2′-O-dimethyl-adenosine (m¹Am), 2′-O-ribosyladenosine(phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine,8-azido-adenosine, 2′-F-ara-adenosine, 2′-F-adenosine,2′-0H-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

In some embodiments, the modified nucleobase is a modified guanine.Exemplary nucleobases and nucleosides having a modified guanine includeinosine (I), 1-methyl-inosine (m¹I), wyosine (imG), methylwyosine(mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW),peroxywybutosine (o2yW), hydroxywybutosine (OhyW), undermodifiedhydroxywybutosine (OhyW*), 7-deaza-guanosine, queuosine (Q),epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine(manQ), 7-cyano-7-deaza-guanosine (preQ₀),7-aminomethyl-7-deaza-guanosine (preQ₁), archaeosine (G⁺),7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m⁷G),6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine,1-methyl-guanosine (m¹G), N2-methyl-guanosine (m²G),N2,N2-dimethyl-guanosine (m² ₂G), N2,7-dimethyl-guano sine (m^(2,7)G),N2, N2,7-dimethyl-guanosine (m^(2,2,7)G), 8-oxo-guanosine,7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine,α-thio-guanosine, 2′-O-methyl-guanosine (Gm),N2-methyl-2′-O-methyl-guanosine (m²Gm),N2,N2-dimethyl-2′-O-methyl-guanosine (m² ₂Gm),1-methyl-2′-O-methyl-guanosine (m¹Gm),N2,7-dimethyl-2′-O-methyl-guanosine (m²′⁷Gm), 2′-O-methyl-inosine (Im),1,2′-O-dimethyl-inosine (m¹Im), 2′-O-ribosylguanosine (phosphate)(Gr(p)), 1-thio-guanosine, 06-methyl-guano sine, 2′-F-ara-guanosine, and2′-F-guanosine.

N-Linked Glycosylation Site Mutants

N-linked glycans of viral proteins play important roles in modulatingthe immune response. Glycans can be important for maintaining theappropriate antigenic conformations, shielding potential neutralizationepitopes, and may alter the proteolytic susceptibility of proteins. Someviruses have putative N-linked glycosylation sites. Deletion ormodification of an N-linked glycosylation site may enhance the immuneresponse. Thus, the present disclosure provides, in some embodiments,RNA (e.g., mRNA) vaccines comprising nucleic acids (e.g., mRNA) encodingantigenic polypeptides that comprise a deletion or modification at oneor more N-linked glycosylation sites.

In Vitro Transcription of RNA (e.g., mRNA)

Tropical disease vaccines of the present disclosure comprise at leastone RNA polynucleotide, such as a mRNA (e.g., modified mRNA). mRNA, forexample, is transcribed in vitro from template DNA, referred to as an“in vitro transcription template.” In some embodiments, an in vitrotranscription template encodes a 5′ untranslated (UTR) region, containsan open reading frame, and encodes a 3′ UTR and a polyA tail. Theparticular nucleic acid sequence composition and length of an in vitrotranscription template will depend on the mRNA encoded by the template.

A “5′ untranslated region” (5′UTR) refers to a region of an mRNA that isdirectly upstream (i.e., 5′) from the start codon (i.e., the first codonof an mRNA transcript translated by a ribosome) that does not encode apolypeptide.

A “3′ untranslated region” (3′UTR) refers to a region of an mRNA that isdirectly downstream (i.e., 3′) from the stop codon (i.e., the codon ofan mRNA transcript that signals a termination of translation) that doesnot encode a polypeptide.

An “open reading frame” is a continuous stretch of DNA beginning with astart codon (e.g., methionine (ATG)), and ending with a stop codon(e.g., TAA, TAG or TGA) and encodes a polypeptide.

A “polyA tail” is a region of mRNA that is downstream, e.g., directlydownstream (i.e., 3′), from the 3′ UTR that contains multiple,consecutive adenosine monophosphates. A polyA tail may contain 10 to 300adenosine monophosphates. For example, a polyA tail may contain 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosinemonophosphates. In some embodiments, a polyA tail contains 50 to 250adenosine monophosphates. In a relevant biological setting (e.g., incells, in vivo) the poly(A) tail functions to protect mRNA fromenzymatic degradation, e.g., in the cytoplasm, and aids in transcriptiontermination, export of the mRNA from the nucleus and translation.

In some embodiments, a polynucleotide includes 200 to 3,000 nucleotides.For example, a polynucleotide may include 200 to 500, 200 to 1000, 200to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to3000 nucleotides.

Flagellin Adjuvants

Flagellin is an approximately 500 amino acid monomeric protein thatpolymerizes to form the flagella associated with bacterial motion.Flagellin is expressed by a variety of flagellated bacteria (Salmonellatyphimurium for example) as well as non-flagellated bacteria (such asEscherichia coli). Sensing of flagellin by cells of the innate immunesystem (dendritic cells, macrophages, etc.) is mediated by the Toll-likereceptor 5 (TLR5) as well as by Nod-like receptors (NLRs) Ipaf andNaip5. TLRs and NLRs have been identified as playing a role in theactivation of innate immune response and adaptive immune response. Assuch, flagellin provides an adjuvant effect in a vaccine.

The nucleotide and amino acid sequences encoding known flagellinpolypeptides are publicly available in the NCBI GenBank database. Theflagellin sequences from S. Typhimurium, H. Pylori, V. Cholera, S.marcesens, S. flexneri, T. pallidum, L. pneumophila, B. burgdorferei, C.difficile, R. meliloti, A. tumefaciens, R. lupini, B. clarridgeiae, P.Mirabilis, B. subtilus, L. monocytogenes, P. aeruginosa, and E. coli,among others are known.

A flagellin polypeptide, as used herein, refers to a full lengthflagellin protein, immunogenic fragments thereof, and peptides having atleast 50% sequence identity to a flagellin protein or immunogenicfragments thereof. Exemplary flagellin proteins include flagellin fromSalmonella typhi (UniPro Entry number: Q56086), Salmonella typhimurium(A0A0C9DG09), Salmonella enteritidis (A0A0C9BAB7), and Salmonellacholeraesuis (Q6V2×8), and proteins having an amino acid sequenceidentified by any one of SEQ ID NO: 420-422 (Table 66). In someembodiments, the flagellin polypeptide has at least 60%, 70%, 75%, 80%,90%, 95%, 97%, 98%, or 99% sequence identity to a flagellin protein orimmunogenic fragments thereof.

In some embodiments, the flagellin polypeptide is an immunogenicfragment. An immunogenic fragment is a portion of a flagellin proteinthat provokes an immune response. In some embodiments, the immuneresponse is a TLR5 immune response. An example of an immunogenicfragment is a flagellin protein in which all or a portion of a hingeregion has been deleted or replaced with other amino acids. For example,an antigenic polypeptide may be inserted in the hinge region. Hingeregions are the hypervariable regions of a flagellin. Hinge regions of aflagellin are also referred to as “D3 domain or region,” “propellerdomain or region,” “hypervariable domain or region” and “variable domainor region.” “At least a portion of a hinge region,” as used herein,refers to any part of the hinge region of the flagellin, or the entiretyof the hinge region. In other embodiments an immunogenic fragment offlagellin is a 20, 25, 30, 35, or 40 amino acid C-terminal fragment offlagellin.

The flagellin monomer is formed by domains D0 through D3. D0 and D1,which form the stem, are composed of tandem long alpha helices and arehighly conserved among different bacteria. The D1 domain includesseveral stretches of amino acids that are useful for TLR5 activation.The entire D1 domain or one or more of the active regions within thedomain are immunogenic fragments of flagellin. Examples of immunogenicregions within the D1 domain include residues 88-114 and residues411-431 in Salmonella typhimurium FliC flagellin. Within the 13 aminoacids in the 88-100 region, at least 6 substitutions are permittedbetween Salmonella flagellin and other flagellins that still preserveTLR5 activation. Thus, immunogenic fragments of flagellin includeflagellin like sequences that activate TLR5 and contain a 13 amino acidmotif that is 53% or more identical to the Salmonella sequence in 88-100of FliC (LQRVRELAVQSAN; SEQ ID NO: 428).

In some embodiments, the RNA (e.g., mRNA) vaccine includes an RNA thatencodes a fusion protein of flagellin and one or more antigenicpolypeptides. A “fusion protein” as used herein, refers to a linking oftwo components of the construct. In some embodiments, a carboxy-terminusof the antigenic polypeptide is fused or linked to an amino terminus ofthe flagellin polypeptide. In other embodiments, an amino-terminus ofthe antigenic polypeptide is fused or linked to a carboxy-terminus ofthe flagellin polypeptide. The fusion protein may include, for example,one, two, three, four, five, six or more flagellin polypeptides linkedto one, two, three, four, five, six or more antigenic polypeptides. Whentwo or more flagellin polypeptides and/or two or more antigenicpolypeptides are linked such a construct may be referred to as a“multimer.”

Each of the components of a fusion protein may be directly linked to oneanother or they may be connected through a linker. For instance, thelinker may be an amino acid linker. The amino acid linker encoded for bythe RNA (e.g., mRNA) vaccine to link the components of the fusionprotein may include, for instance, at least one member selected from thegroup consisting of a lysine residue, a glutamic acid residue, a serineresidue and an arginine residue. In some embodiments the linker is 1-30,1-25, 1-25, 5-10, 5, 15, or 5-20 amino acids in length.

In other embodiments the RNA (e.g., mRNA) vaccine includes at least twoseparate RNA polynucleotides, one encoding one or more antigenicpolypeptides and the other encoding the flagellin polypeptide. The atleast two RNA polynucleotides may be coformulated in a carrier such as alipid nanoparticle.

Broad Spectrum RNA (e.g., mRNA) Vaccines

There may be situations where persons are at risk for infection withmore than one strain of Malaria (e.g., P. falciparum, P. vivax, P.malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKVand/or YFV. RNA (e.g., mRNA) therapeutic vaccines are particularlyamenable to combination vaccination approaches due to a number offactors including, but not limited to, speed of manufacture, ability torapidly tailor vaccines to accommodate perceived geographical threat,and the like. Moreover, because the vaccines utilize the human body toproduce the antigenic protein, the vaccines are amenable to theproduction of larger, more complex antigenic proteins, allowing forproper folding, surface expression, antigen presentation, etc. in thehuman subject. To protect against more than one strain of Malaria (e.g.,P. falciparum, P. vivax, P. malariae and/or P. ovale), JEV, WNV, EEEV,VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV, a combination vaccine can beadministered that includes RNA (e.g., mRNA) encoding at least oneantigenic polypeptide protein (or antigenic portion thereof) of a firsttropical disease virus or organism and further includes RNA encoding atleast one antigenic polypeptide protein (or antigenic portion thereof)of a second tropical disease virus or organism. RNA (e.g., mRNA) can becoformulated, for example, in a single lipid nanoparticle (LNP) or canbe formulated in separate LNPs for co-administration.

Methods of Treatment

Provided herein are compositions (e.g., pharmaceutical compositions),methods, kits and reagents for prevention and/or treatment of tropicaldiseases in humans and other mammals. Tropical disease RNA (e.g. mRNA)vaccines can be used as therapeutic or prophylactic agents, alone or incombination with other vaccine(s). They may be used in medicine toprevent and/or treat tropical disease. In exemplary aspects, the RNA(e.g., mRNA) vaccines of the present disclosure are used to provideprophylactic protection from Malaria (e.g., P. falciparum, P. vivax, P.malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKVand/or YFV. Prophylactic protection from Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV,CHIKV, DENV, ZIKV and/or YFV can be achieved following administration ofa RNA (e.g., mRNA) vaccine of the present disclosure. Tropical diseaseRNA (e.g., mRNA) vaccines of the present disclosure may be used to treator prevent viral “co-infections” containing two or more tropical diseaseinfections. Vaccines can be administered once, twice, three times, fourtimes or more, but it is likely sufficient to administer the vaccineonce (optionally followed by a single booster). It is possible, althoughless desirable, to administer the vaccine to an infected individual toachieve a therapeutic response. Dosing may need to be adjustedaccordingly.

A method of eliciting an immune response in a subject against Malaria(e.g., P. falciparum, P. vivax, P. malariae and/or P. ovale), JEV, WNV,EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV is provided in aspects ofthe present disclosure. The method involves administering to the subjecta tropical disease RNA (e.g., mRNA) vaccine comprising at least one RNA(e.g., mRNA) polynucleotide having an open reading frame encoding atleast one Malaria (e.g., P. falciparum, P. vivax, P. malariae and/or P.ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFVantigenic polypeptide, thereby inducing in the subject an immuneresponse specific to Malaria (e.g., P. falciparum, P. vivax, P. malariaeand/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/orYFV antigenic polypeptide or an immunogenic fragment thereof, whereinanti-antigenic polypeptide antibody titer in the subject is increasedfollowing vaccination relative to anti-antigenic polypeptide antibodytiter in a subject vaccinated with a prophylactically effective dose ofa traditional vaccine against Malaria (e.g., P. falciparum, P. vivax, P.malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKVand/or YFV. An “anti-antigenic polypeptide antibody” is a serum antibodythe binds specifically to the antigenic polypeptide.

In some embodiments, a RNA (e.g., mRNA) vaccine (e.g., a Malaria (e.g.,P. falciparum, P. vivax, P. malariae and/or P. ovale), JEV, WNV, EEEV,VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV RNA vaccine) capable ofeliciting an immune response is administered intramuscularly orintranasally via a composition including a compound according to Formula(I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe) (e.g., Compound 3,18, 20, 25, 26, 29, 30, 60, 108-112, or 122).

A prophylactically effective dose is a therapeutically effective dosethat prevents infection with the virus at a clinically acceptable level.In some embodiments the therapeutically effective dose is a dose listedin a package insert for the vaccine. A traditional vaccine, as usedherein, refers to a vaccine other than the RNA (e.g., mRNA) vaccines ofthe present disclosure. For instance, a traditional vaccine includes butis not limited to live/attenuated microorganism vaccines,killed/inactivated microorganism vaccines, subunit vaccines, proteinantigen vaccines, DNA vaccines, VLP vaccines, etc. In exemplaryembodiments, a traditional vaccine is a vaccine that has achievedregulatory approval and/or is registered by a national drug regulatorybody, for example the Food and Drug Administration (FDA) in the UnitedStates or the European Medicines Agency (EMA).

In some embodiments the anti-antigenic polypeptide antibody titer in thesubject is increased 1 log to 10 log following vaccination relative toanti-antigenic polypeptide antibody titer in a subject vaccinated with aprophylactically effective dose of a traditional vaccine against Malaria(e.g., P. falciparum, P. vivax, P. malariae and/or P. ovale), JEV, WNV,EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV.

In some embodiments the anti-antigenic polypeptide antibody titer in thesubject is increased 1 log, 2 log, 3 log, 5 log or 10 log followingvaccination relative to anti-antigenic polypeptide antibody titer in asubject vaccinated with a prophylactically effective dose of atraditional vaccine against Malaria (e.g., P. falciparum, P. vivax, P.malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKVand/or YFV.

A method of eliciting an immune response in a subject against Malaria(e.g., P. falciparum, P. vivax, P. malariae and/or P. ovale), JEV, WNV,EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV is provided in otheraspects of the disclosure. The method involves administering to thesubject a tropical disease RNA (e.g., mRNA) vaccine comprising at leastone RNA (e.g., mRNA) polynucleotide having an open reading frameencoding at least one Malaria (e.g., P. falciparum, P. vivax, P.malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKVand/or YFV antigenic polypeptide or an immunogenic fragment thereof,thereby inducing in the subject an immune response specific to Malaria(e.g., P. falciparum, P. vivax, P. malariae and/or P. ovale), JEV, WNV,EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV antigenic polypeptide oran immunogenic fragment thereof, wherein the immune response in thesubject is equivalent to an immune response in a subject vaccinated witha traditional vaccine against the Malaria (e.g., P. falciparum, P.vivax, P. malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV,DENV, ZIKV and/or YFV at 2 times to 100 times the dosage level relativeto the RNA (e.g., mRNA) vaccine.

In some embodiments, the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine at2, 3, 4, 5, 10, 50, 100 times the dosage level relative to the Malaria(e.g., P. falciparum, P. vivax, P. malariae and/or P. ovale), JEV, WNV,EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV RNA (e.g., mRNA) vaccine.

In some embodiments the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine at10-100 times, or 100-1000 times, the dosage level relative to theMalaria (e.g., P. falciparum, P. vivax, P. malariae and/or P. ovale),JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV RNA (e.g.,mRNA) vaccine.

In some embodiments the immune response is assessed by determiningprotein antibody titer in the subject.

Some embodiments provide a method of inducing an immune response in asubject by administering to the subject a tropical disease RNA (e.g.,mRNA) vaccine comprising at least one RNA (e.g., mRNA) polynucleotidehaving an open reading frame encoding at least one Malaria (e.g., P.falciparum, P. vivax, P. malariae and/or P. ovale), JEV, WNV, EEEV,VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV antigenic polypeptide, therebyinducing in the subject an immune response specific to the antigenicpolypeptide or an immunogenic fragment thereof, wherein the immuneresponse in the subject is induced 2 days to 10 weeks earlier relativeto an immune response induced in a subject vaccinated with aprophylactically effective dose of a traditional vaccine against Malaria(e.g., P. falciparum, P. vivax, P. malariae and/or P. ovale), JEV, WNV,EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV. In some embodiments, theimmune response in the subject is induced in a subject vaccinated with aprophylactically effective dose of a traditional vaccine at 2 times to100 times the dosage level relative to the RNA (e.g., mRNA) vaccine.

In some embodiments the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine at2, 3, 4, 5, 10, 50, 100 times the dosage level relative to the Malaria(e.g., P. falciparum, P. vivax, P. malariae and/or P. ovale), JEV, WNV,EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV RNA (e.g., mRNA) vaccine.

In some embodiments, the immune response in the subject is induced 2days earlier, or 3 days earlier, relative to an immune response inducedin a subject vaccinated with a prophylactically effective dose of atraditional vaccine.

In some embodiments the immune response in the subject is induced 1week, 2 weeks, 3 weeks, 5 weeks, or 10 weeks earlier relative to animmune response induced in a subject vaccinated with a prophylacticallyeffective dose of a traditional vaccine.

Therapeutic and Prophylactic Compositions

Provided herein are compositions (e.g., pharmaceutical compositions),methods, kits and reagents for prevention, treatment or diagnosis ofMalaria (e.g., P. falciparum, P. vivax, P. malariae and/or P. ovale),JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV in humans andother mammals, for example. Tropical disease RNA (e.g. mRNA) vaccinescan be used as therapeutic or prophylactic agents. They may be used inmedicine to prevent and/or treat infectious disease. In someembodiments, the RNA (e.g., mRNA) vaccines of the present disclosure areused fin the priming of immune effector cells, for example, to activateperipheral blood mononuclear cells (PBMCs) ex vivo, which are theninfused (re-infused) into a subject.

In some embodiments, tropical disease vaccine containing RNA (e.g.,mRNA) polynucleotides as described herein can be administered to asubject (e.g., a mammalian subject, such as a human subject), and theRNA (e.g., mRNA) polynucleotides are translated in vivo to produce anantigenic polypeptide.

The tropical disease RNA (e.g., mRNA) vaccines may be induced fortranslation of a polypeptide (e.g., antigen or immunogen) in a cell,tissue or organism. In some embodiments, such translation occurs invivo, although such translation may occur ex vivo, in culture or invitro. In some embodiments, the cell, tissue or organism is contactedwith an effective amount of a composition containing a tropical diseaseRNA (e.g., mRNA) vaccine that contains a polynucleotide that has atleast one a translatable region encoding an antigenic polypeptide.

An “effective amount” of a tropical disease RNA (e.g. mRNA) vaccine isprovided based, at least in part, on the target tissue, target celltype, means of administration, physical characteristics of thepolynucleotide (e.g., size, and extent of modified nucleosides) andother components of the vaccine, and other determinants. In general, aneffective amount of the tropical disease RNA (e.g., mRNA) vaccinecomposition provides an induced or boosted immune response as a functionof antigen production in the cell, preferably more efficient than acomposition containing a corresponding unmodified polynucleotideencoding the same antigen or a peptide antigen. Increased antigenproduction may be demonstrated by increased cell transfection (thepercentage of cells transfected with the RNA, e.g., mRNA, vaccine),increased protein translation from the polynucleotide, decreased nucleicacid degradation (as demonstrated, for example, by increased duration ofprotein translation from a modified polynucleotide), or altered antigenspecific immune response of the host cell.

In some embodiments, RNA (e.g. mRNA) vaccines (including polynucleotidestheir encoded polypeptides) in accordance with the present disclosuremay be used for treatment of Malaria (e.g., P. falciparum, P. vivax, P.malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKVand/or YFV.

Tropical disease RNA (e.g. mRNA) vaccines may be administeredprophylactically or therapeutically as part of an active immunizationscheme to healthy individuals or early in infection during theincubation phase or during active infection after onset of symptoms. Insome embodiments, the amount of RNA (e.g., mRNA) vaccine of the presentdisclosure provided to a cell, a tissue or a subject may be an amounteffective for immune prophylaxis.

Tropical disease RNA (e.g. mRNA) vaccines may be administrated withother prophylactic or therapeutic compounds. As a non-limiting example,a prophylactic or therapeutic compound may be an adjuvant or a booster.As used herein, when referring to a prophylactic composition, such as avaccine, the term “booster” refers to an extra administration of theprophylactic (vaccine) composition. A booster (or booster vaccine) maybe given after an earlier administration of the prophylacticcomposition. The time of administration between the initialadministration of the prophylactic composition and the booster may be,but is not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15minutes, 20 minutes 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, 5 days, 6days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years,7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95years or more than 99 years. In some embodiments, the time ofadministration between the initial administration of the prophylacticcomposition and the booster may be, but is not limited to, 1 week, 2weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months or 1 year.

In some embodiments, tropical disease RNA (e.g. mRNA) vaccines may beadministered intramuscularly, intradermally, or intranasally, similarlyto the administration of inactivated vaccines known in the art.

Tropical disease RNA (e.g. mRNA) vaccines may be utilized in varioussettings depending on the prevalence of the infection or the degree orlevel of unmet medical need. As a non-limiting example, the RNA (e.g.,mRNA) vaccines may be utilized to treat and/or prevent a variety oftropical diseases. RNA (e.g., mRNA) vaccines have superior properties inthat they produce much larger antibody titers and produce responsesearly than commercially available anti-viral agents/compositions.

Provided herein are pharmaceutical compositions including tropicaldisease RNA (e.g. mRNA) vaccines and RNA (e.g. mRNA) vaccinecompositions and/or complexes optionally in combination with one or morepharmaceutically acceptable excipients.

Malaria (e.g., P. falciparum, P. vivax, P. malariae and/or P. ovale),JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV RNA (e.g. mRNA)vaccines may be formulated or administered alone or in conjunction withone or more other components. For instance, Malaria (e.g., P.falciparum, P. vivax, P. malariae and/or P. ovale), JEV, WNV, EEEV,VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV RNA (e.g., mRNA) vaccines(vaccine compositions) may comprise other components including, but notlimited to, adjuvants.

In some embodiments, tropical disease (e.g. mRNA) vaccines do notinclude an adjuvant (they are adjuvant free).

Tropical disease RNA (e.g. mRNA) vaccines may be formulated oradministered in combination with one or more pharmaceutically-acceptableexcipients. In some embodiments, vaccine compositions comprise at leastone additional active substances, such as, for example, atherapeutically-active substance, a prophylactically-active substance,or a combination of both. Vaccine compositions may be sterile,pyrogen-free or both sterile and pyrogen-free. General considerations inthe formulation and/or manufacture of pharmaceutical agents, such asvaccine compositions, may be found, for example, in Remington: TheScience and Practice of Pharmacy 21st ed., Lippincott Williams &Wilkins, 2005 (incorporated herein by reference in its entirety).

In some embodiments, tropical disease RNA (e.g. mRNA) vaccines areadministered to humans, human patients or subjects. For the purposes ofthe present disclosure, the phrase “active ingredient” generally refersto the RNA (e.g., mRNA) vaccines or the polynucleotides containedtherein, for example, RNA polynucleotides (e.g., mRNA polynucleotides)encoding antigenic polypeptides.

Formulations of the tropical disease vaccine compositions describedherein may be prepared by any method known or hereafter developed in theart of pharmacology. In general, such preparatory methods include thestep of bringing the active ingredient (e.g., mRNA polynucleotide) intoassociation with an excipient and/or one or more other accessoryingredients, and then, if necessary and/or desirable, dividing, shapingand/or packaging the product into a desired single- or multi-dose unit.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the disclosure will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered. By way of example, the composition may comprisebetween 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between5-80%, at least 80% (w/w) active ingredient.

Tropical disease RNA (e.g. mRNA) vaccines can be formulated using one ormore excipients to: increase stability; increase cell transfection;permit the sustained or delayed release (e.g., from a depotformulation); alter the biodistribution (e.g., target to specifictissues or cell types); increase the translation of encoded protein invivo; and/or alter the release profile of encoded protein (antigen) invivo. In addition to traditional excipients such as any and allsolvents, dispersion media, diluents, or other liquid vehicles,dispersion or suspension aids, surface active agents, isotonic agents,thickening or emulsifying agents, preservatives, excipients can include,without limitation, lipidoids, liposomes, lipid nanoparticles, polymers,lipoplexes, core-shell nanoparticles, peptides, proteins, cellstransfected with tropical disease RNA (e.g. mRNA) vaccines (e.g., fortransplantation into a subject), hyaluronidase, nanoparticle mimics andcombinations thereof.

Stabilizing Elements

Naturally-occurring eukaryotic mRNA molecules have been found to containstabilizing elements, including, but not limited to untranslated regions(UTR) at their 5′-end (5′UTR) and/or at their 3′-end (3′UTR), inaddition to other structural features, such as a 5′-cap structure or a3′-poly(A) tail. Both the 5′UTR and the 3′UTR are typically transcribedfrom the genomic DNA and are elements of the premature mRNA.Characteristic structural features of mature mRNA, such as the 5′-capand the 3′-poly(A) tail are usually added to the transcribed (premature)mRNA during mRNA processing. The 3′-poly(A) tail is typically a stretchof adenine nucleotides added to the 3′-end of the transcribed mRNA. Itcan comprise up to about 400 adenine nucleotides. In some embodimentsthe length of the 3′-poly(A) tail may be an essential element withrespect to the stability of the individual mRNA.

In some embodiments the RNA (e.g., mRNA) vaccine may include one or morestabilizing elements. Stabilizing elements may include for instance ahistone stem-loop. A stem-loop binding protein (SLBP), a 32 kDa proteinhas been identified. It is associated with the histone stem-loop at the3′-end of the histone messages in both the nucleus and the cytoplasm.Its expression level is regulated by the cell cycle; it peaks during theS-phase, when histone mRNA levels are also elevated. The protein hasbeen shown to be essential for efficient 3′-end processing of histonepre-mRNA by the U7 snRNP. SLBP continues to be associated with thestem-loop after processing, and then stimulates the translation ofmature histone mRNAs into histone proteins in the cytoplasm. The RNAbinding domain of SLBP is conserved through metazoa and protozoa; itsbinding to the histone stem-loop depends on the structure of the loop.The minimum binding site includes at least three nucleotides 5′ and twonucleotides 3′ relative to the stem-loop.

In some embodiments, the RNA (e.g., mRNA) vaccines include a codingregion, at least one histone stem-loop, and optionally, a poly(A)sequence or polyadenylation signal. The poly(A) sequence orpolyadenylation signal generally should enhance the expression level ofthe encoded protein. The encoded protein, in some embodiments, is not ahistone protein, a reporter protein (e.g. Luciferase, GFP, EGFP,β-Galactosidase, EGFP), or a marker or selection protein (e.g.alpha-Globin, Galactokinase and Xanthine:guanine phosphoribosyltransferase (GPT)).

In some embodiments, the combination of a poly(A) sequence orpolyadenylation signal and at least one histone stem-loop, even thoughboth represent alternative mechanisms in nature, acts synergistically toincrease the protein expression beyond the level observed with either ofthe individual elements. It has been found that the synergistic effectof the combination of poly(A) and at least one histone stem-loop doesnot depend on the order of the elements or the length of the poly(A)sequence.

In some embodiments, the RNA (e.g., mRNA) vaccine does not comprise ahistone downstream element (HDE). “Histone downstream element” (HDE)includes a purine-rich polynucleotide stretch of approximately 15 to 20nucleotides 3′ of naturally occurring stem-loops, representing thebinding site for the U7 snRNA, which is involved in processing ofhistone pre-mRNA into mature histone mRNA. Ideally, the inventivenucleic acid does not include an intron.

In some embodiments, the RNA (e.g., mRNA) vaccine may or may not containan enhancer and/or promoter sequence, which may be modified orunmodified or which may be activated or inactivated. In someembodiments, the histone stem-loop is generally derived from histonegenes, and includes an intramolecular base pairing of two neighboredpartially or entirely reverse complementary sequences separated by aspacer, including (e.g., consisting of) a short sequence, which formsthe loop of the structure. The unpaired loop region is typically unableto base pair with either of the stem loop elements. It occurs more oftenin RNA, as is a key component of many RNA secondary structures, but maybe present in single-stranded DNA as well. Stability of the stem-loopstructure generally depends on the length, number of mismatches orbulges, and base composition of the paired region. In some embodiments,wobble base pairing (non-Watson-Crick base pairing) may result. In someembodiments, the at least one histone stem-loop sequence comprises alength of 15 to 45 nucleotides.

In other embodiments the RNA (e.g., mRNA) vaccine may have one or moreAU-rich sequences removed. These sequences, sometimes referred to asAURES, are destabilizing sequences found in the 3′UTR. The AURES may beremoved from the RNA (e.g., mRNA) vaccines. Alternatively the AURES mayremain in the RNA (e.g., mRNA) vaccine.

Nanoparticle Formulations

In some embodiments, tropical disease RNA (e.g. mRNA) vaccines areformulated in a nanoparticle. In some embodiments, tropical disease RNA(e.g. mRNA) vaccines are formulated in a lipid nanoparticle. In someembodiments, tropical disease RNA (e.g. mRNA) vaccines are formulated ina lipid-polycation complex, referred to as a cationic lipidnanoparticle. As a non-limiting example, the polycation may include acationic peptide or a polypeptide such as, but not limited to,polylysine, polyornithine and/or polyarginine. In some embodiments,tropical disease RNA (e.g., mRNA) vaccines are formulated in a lipidnanoparticle that includes a non-cationic lipid such as, but not limitedto, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).

A lipid nanoparticle formulation may be influenced by, but not limitedto, the selection of the cationic lipid component, the degree ofcationic lipid saturation, the nature of the PEGylation, ratio of allcomponents and biophysical parameters such as size. In one example bySemple et al. (Nature Biotech. 2010 28:172-176), the lipid nanoparticleformulation is composed of 57.1% cationic lipid, 7.1%dipalmitoylphosphatidylcholine, 34.3% cholesterol, and 1.4% PEG-c-DMA.As another example, changing the composition of the cationic lipid canmore effectively deliver siRNA to various antigen presenting cells(Basha et al. Mol Ther. 2011 19:2186-2200).

In some embodiments, lipid nanoparticle formulations may comprise 35 to45% cationic lipid, 40% to 50% cationic lipid, 50% to 60% cationic lipidand/or 55% to 65% cationic lipid. In some embodiments, the ratio oflipid to RNA (e.g., mRNA) in lipid nanoparticles may be 5:1 to 20:1,10:1 to 25:1, 15:1 to 30:1 and/or at least 30:1.

In some embodiments, the ratio of PEG in the lipid nanoparticleformulations may be increased or decreased and/or the carbon chainlength of the PEG lipid may be modified from C14 to C18 to alter thepharmacokinetics and/or biodistribution of the lipid nanoparticleformulations. As a non-limiting example, lipid nanoparticle formulationsmay contain 0.5% to 3.0%, 1.0% to 3.5%, 1.5% to 4.0%, 2.0% to 4.5%, 2.5%to 5.0% and/or 3.0% to 6.0% of the lipid molar ratio of PEG-c-DOMG(R-3-[(ω-methoxy-poly(ethyleneglycol)2000)carbamoyl)]-1,2-dimyristyloxypropyl-3-amine)(also referred to herein as PEG-DOMG) as compared to the cationic lipid,DSPC and cholesterol. In some embodiments, the PEG-c-DOMG may bereplaced with a PEG lipid such as, but not limited to, PEG-DSG(1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG(1,2-Dimyristoyl-sn-glycerol) and/or PEG-DPG(1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The cationiclipid may be selected from any lipid known in the art such as, but notlimited to, DLin-MC3-DMA, DLin-DMA, C12-200 and DLin-KC2-DMA.

In some embodiments, a tropical disease RNA (e.g. mRNA) vaccineformulation is a nanoparticle that comprises at least one lipid. Thelipid may be selected from, but is not limited to, DLin-DMA, DLin-K-DMA,98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG,PEGylated lipids and amino alcohol lipids. In some embodiments, thelipid may be a cationic lipid such as, but not limited to, DLin-DMA,DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA and amino alcohol lipids.The amino alcohol cationic lipid may be the lipids described in and/ormade by the methods described in U.S. Patent Publication No.US20130150625, herein incorporated by reference in its entirety. As anon-limiting example, the cationic lipid may be2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol(Compound 1 in US20130150625);2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol(Compound 2 in US20130150625);2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol(Compound 3 in US20130150625); and2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol(Compound 4 in US20130150625); or any pharmaceutically acceptable saltor stereoisomer thereof.

Lipid nanoparticle formulations typically comprise a lipid, inparticular, an ionizable cationic lipid, for example,2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), ordi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), and furthercomprise a neutral lipid, a sterol and a molecule capable of reducingparticle aggregation, for example a PEG or PEG-modified lipid.

In some embodiments, a lipid nanoparticle formulation consistsessentially of (i) at least one lipid selected from the group consistingof 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); (ii) aneutral lipid selected from DSPC, DPPC, POPC, DOPE and SM; (iii) asterol, e.g., cholesterol; and (iv) a PEG-lipid, e.g., PEG-DMG orPEG-cDMA, in a molar ratio of 20-60% cationic lipid:5-25% neutrallipid:25-55% sterol; 0.5-15% PEG-lipid.

In some embodiments, a lipid nanoparticle formulation includes 25% to75% on a molar basis of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g., 35% to65%, 45% to 65%, 60%, 57.5%, 50% or 40% on a molar basis.

In some embodiments, a lipid nanoparticle formulation includes 0.5% to15% on a molar basis of the neutral lipid, e.g., 3% to 12%, 5% to 10% or15%, 10%, or 7.5% on a molar basis. Examples of neutral lipids include,without limitation, DSPC, POPC, DPPC, DOPE and SM. In some embodiments,the formulation includes 5% to 50% on a molar basis of the sterol (e.g.,15% to 45%, 20% to 40%, 40%, 38.5%, 35%, or 31% on a molar basis). Anon-limiting example of a sterol is cholesterol. In some embodiments, alipid nanoparticle formulation includes 0.5% to 20% on a molar basis ofthe PEG or PEG-modified lipid (e.g., 0.5% to 10%, 0.5% to 5%, 1.5%,0.5%, 1.5%, 3.5%, or 5% on a molar basis). In some embodiments, a PEG orPEG modified lipid comprises a PEG molecule of an average molecularweight of 2,000 Da. In some embodiments, a PEG or PEG modified lipidcomprises a PEG molecule of an average molecular weight of less than2,000, for example around 1,500 Da, around 1,000 Da, or around 500 Da.Non-limiting examples of PEG-modified lipids include PEG-distearoylglycerol (PEG-DMG) (also referred herein as PEG-C14 or C14-PEG),PEG-cDMA (further discussed in Reyes et al. J. Controlled Release, 107,276-287 (2005) the contents of which are herein incorporated byreference in their entirety).

In some embodiments, lipid nanoparticle formulations include 25-75% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 0.5-15% ofthe neutral lipid, 5-50% of the sterol, and 0.5-20% of the PEG orPEG-modified lipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 35-65% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 3-12% of theneutral lipid, 15-45% of the sterol, and 0.5-10% of the PEG orPEG-modified lipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 45-65% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 5-10% of theneutral lipid, 25-40% of the sterol, and 0.5-10% of the PEG orPEG-modified lipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 60% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 7.5% of theneutral lipid, 31% of the sterol, and 1.5% of the PEG or PEG-modifiedlipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 50% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 10% of theneutral lipid, 38.5% of the sterol, and 1.5% of the PEG or PEG-modifiedlipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 50% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 10% of theneutral lipid, 35% of the sterol, 4.5% or 5% of the PEG or PEG-modifiedlipid, and 0.5% of the targeting lipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 40% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 15% of theneutral lipid, 40% of the sterol, and 5% of the PEG or PEG-modifiedlipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 57.2% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 7.1% of theneutral lipid, 34.3% of the sterol, and 1.4% of the PEG or PEG-modifiedlipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 57.5% of acationic lipid selected from the PEG lipid is PEG-cDMA (PEG-cDMA isfurther discussed in Reyes et al. (J. Controlled Release, 107, 276-287(2005), the contents of which are herein incorporated by reference intheir entirety), 7.5% of the neutral lipid, 31.5% of the sterol, and3.5% of the PEG or PEG-modified lipid on a molar basis.

In some embodiments, lipid nanoparticle formulations consist essentiallyof a lipid mixture in molar ratios of 20-70% cationic lipid:5-45%neutral lipid:20-55% cholesterol:0.5-15% PEG-modified lipid. In someembodiments, lipid nanoparticle formulations consist essentially of alipid mixture in a molar ratio of 20-60% cationic lipid:5-25% neutrallipid: 25-55% cholesterol:0.5-15% PEG-modified lipid.

In some embodiments, the molar lipid ratio is 50/10/38.5/1.5 (mol %cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g.,PEG-DMG, PEG-DSG or PEG-DPG), 57.2/7.1134.3/1.4 (mol % cationiclipid/neutral lipid, e.g., DPPC/Chol/PEG-modified lipid, e.g.,PEG-cDMA), 40/15/40/5 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG), 50/10/35/4.5/0.5 (mol %cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g.,PEG-DSG), 50/10/35/5 (cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG), 40/10/40/10 (mol %cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g.,PEG-DMG or PEG-cDMA), 35/15/40/10 (mol % cationic lipid/neutral lipid,e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA) or52/13/30/5 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA).

Non-limiting examples of lipid nanoparticle compositions and methods ofmaking them are described, for example, in Semple et al. (2010) Nat.Biotechnol. 28:172-176; Jayarama et al. (2012), Angew. Chem. Int. Ed.,51: 8529-8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578(the contents of each of which are incorporated herein by reference intheir entirety).

In some embodiments, lipid nanoparticle formulations may comprise acationic lipid, a PEG lipid and a structural lipid and optionallycomprise a non-cationic lipid. As a non-limiting example, a lipidnanoparticle may comprise 40-60% of cationic lipid, 5-15% of anon-cationic lipid, 1-2% of a PEG lipid and 30-50% of a structurallipid. As another non-limiting example, the lipid nanoparticle maycomprise 50% cationic lipid, 10% non-cationic lipid, 1.5% PEG lipid and38.5% structural lipid. As yet another non-limiting example, a lipidnanoparticle may comprise 55% cationic lipid, 10% non-cationic lipid,2.5% PEG lipid and 32.5% structural lipid. In some embodiments, thecationic lipid may be any cationic lipid described herein such as, butnot limited to, DLin-KC2-DMA, DLin-MC3-DMA and L319.

In some embodiments, the lipid nanoparticle formulations describedherein may be 4 component lipid nanoparticles. The lipid nanoparticlemay comprise a cationic lipid, a non-cationic lipid, a PEG lipid and astructural lipid. As a non-limiting example, the lipid nanoparticle maycomprise 40-60% of cationic lipid, 5-15% of a non-cationic lipid, 1-2%of a PEG lipid and 30-50% of a structural lipid. As another non-limitingexample, the lipid nanoparticle may comprise 50% cationic lipid, 10%non-cationic lipid, 1.5% PEG lipid and 38.5% structural lipid. As yetanother non-limiting example, the lipid nanoparticle may comprise 55%cationic lipid, 10% non-cationic lipid, 2.5% PEG lipid and 32.5%structural lipid. In some embodiments, the cationic lipid may be anycationic lipid described herein such as, but not limited to,DLin-KC2-DMA, DLin-MC3-DMA and L319.

In some embodiments, the lipid nanoparticle formulations describedherein may comprise a cationic lipid, a non-cationic lipid, a PEG lipidand a structural lipid. As a non-limiting example, the lipidnanoparticle comprises 50% of the cationic lipid DLin-KC2-DMA, 10% ofthe non-cationic lipid DSPC, 1.5% of the PEG lipid PEG-DOMG and 38.5% ofthe structural lipid cholesterol. As a non-limiting example, the lipidnanoparticle comprises 50% of the cationic lipid DLin-MC3-DMA, 10% ofthe non-cationic lipid DSPC, 1.5% of the PEG lipid PEG-DOMG and 38.5% ofthe structural lipid cholesterol. As a non-limiting example, the lipidnanoparticle comprises 50% of the cationic lipid DLin-MC3-DMA, 10% ofthe non-cationic lipid DSPC, 1.5% of the PEG lipid PEG-DMG and 38.5% ofthe structural lipid cholesterol. As yet another non-limiting example,the lipid nanoparticle comprises 55% of the cationic lipid L319, 10% ofthe non-cationic lipid DSPC, 2.5% of the PEG lipid PEG-DMG and 32.5% ofthe structural lipid cholesterol.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in a vaccinecomposition may vary, depending upon the identity, size, and/orcondition of the subject being treated and further depending upon theroute by which the composition is to be administered. For example, thecomposition may comprise between 0.1% and 99% (w/w) of the activeingredient. By way of example, the composition may comprise between 0.1%and 100%, e.g., between 0.5% and 50%, between 1-30%, between 5-80%, atleast 80% (w/w) active ingredient.

In some embodiments, the tropical disease RNA (e.g. mRNA) vaccinecomposition may comprise the polynucleotide described herein, formulatedin a lipid nanoparticle comprising MC3, Cholesterol, DSPC andPEG2000-DMG, the buffer trisodium citrate, sucrose and water forinjection. As a non-limiting example, the composition comprises: 2.0mg/mL of drug substance, 21.8 mg/mL of MC3, 10.1 mg/mL of cholesterol,5.4 mg/mL of DSPC, 2.7 mg/mL of PEG2000-DMG, 5.16 mg/mL of trisodiumcitrate, 71 mg/mL of sucrose and 1.0 mL of water for injection.

In some embodiments, a nanoparticle (e.g., a lipid nanoparticle) has amean diameter of 10-500 nm, 20-400 nm, 30-300 nm, or 40-200 nm. In someembodiments, a nanoparticle (e.g., a lipid nanoparticle) has a meandiameter of 50-150 nm, 50-200 nm, 80-100 nm or 80-200 nm.

Liposomes, Lipoplexes, and Lipid Nanoparticles

The RNA (e.g., mRNA) vaccines of the disclosure can be formulated usingone or more liposomes, lipoplexes, or lipid nanoparticles. In someembodiments, pharmaceutical compositions of RNA (e.g., mRNA) vaccinesinclude liposomes. Liposomes are artificially-prepared vesicles whichmay primarily be composed of a lipid bilayer and may be used as adelivery vehicle for the administration of nutrients and pharmaceuticalformulations. Liposomes can be of different sizes such as, but notlimited to, a multilamellar vesicle (MLV) which may be hundreds ofnanometers in diameter and may contain a series of concentric bilayersseparated by narrow aqueous compartments, a small unicellular vesicle(SUV) which may be smaller than 50 nm in diameter, and a largeunilamellar vesicle (LUV) which may be between 50 and 500 nm indiameter. Liposome design may include, but is not limited to, opsoninsor ligands in order to improve the attachment of liposomes to unhealthytissue or to activate events such as, but not limited to, endocytosis.Liposomes may contain a low or a high pH in order to improve thedelivery of the pharmaceutical formulations.

The formation of liposomes may depend on the physicochemicalcharacteristics such as, but not limited to, the pharmaceuticalformulation entrapped and the liposomal ingredients, the nature of themedium in which the lipid vesicles are dispersed, the effectiveconcentration of the entrapped substance and its potential toxicity, anyadditional processes involved during the application and/or delivery ofthe vesicles, the optimization size, polydispersity and the shelf-lifeof the vesicles for the intended application, and the batch-to-batchreproducibility and possibility of large-scale production of safe andefficient liposomal products.

In some embodiments, pharmaceutical compositions described herein mayinclude, without limitation, liposomes such as those formed from1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2liposomes from Marina Biotech (Bothell, Wash.),1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA),2,2-dilinoleyl-4-(2-dimethylaminoethyl)[1,3]-dioxolane (DLin-KC2-DMA),and MC3 (US20100324120; herein incorporated by reference in itsentirety) and liposomes which may deliver small molecule drugs such as,but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, Pa.).

In some embodiments, pharmaceutical compositions described herein mayinclude, without limitation, liposomes such as those formed from thesynthesis of stabilized plasmid-lipid particles (SPLP) or stabilizednucleic acid lipid particle (SNALP) that have been previously describedand shown to be suitable for oligonucleotide delivery in vitro and invivo (see Wheeler et al. Gene Therapy. 1999 6:271-281; Zhang et al. GeneTherapy. 1999 6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372;Morrissey et al., Nat Biotechnol. 2005 2:1002-1007; Zimmermann et al.,Nature. 2006 441:111-114; Heyes et al. J Contr Rel. 2005 107:276-287;Semple et al. Nature Biotech. 2010 28:172-176; Judge et al. J ClinInvest. 2009 119:661-673; deFougerolles Hum Gene Ther. 2008 19:125-132;U.S. Patent Publication No US20130122104; all of which are incorporatedherein in their entireties). The original manufacture method by Wheeleret al. was a detergent dialysis method, which was later improved byJeffs et al. and is referred to as the spontaneous vesicle formationmethod. The liposome formulations are composed of 3 to 4 lipidcomponents in addition to the polynucleotide. As an example a liposomecan contain, but is not limited to, 55% cholesterol, 20%disteroylphosphatidyl choline (DSPC), 10% PEG-S-DSG, and 15%1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), as described by Jeffset al. As another example, certain liposome formulations may contain,but are not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30%cationic lipid, where the cationic lipid can be1,2-distearloxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or1,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA), as described byHeyes et al.

In some embodiments, liposome formulations may comprise from about 25.0%cholesterol to about 40.0% cholesterol, from about 30.0% cholesterol toabout 45.0% cholesterol, from about 35.0% cholesterol to about 50.0%cholesterol and/or from about 48.5% cholesterol to about 60%cholesterol. In some embodiments, formulations may comprise a percentageof cholesterol selected from the group consisting of 28.5%, 31.5%,33.5%, 36.5%, 37.0%, 38.5%, 39.0% and 43.5%. In some embodiments,formulations may comprise from about 5.0% to about 10.0% DSPC and/orfrom about 7.0% to about 15.0% DSPC.

In some embodiments, the RNA (e.g., mRNA) vaccine pharmaceuticalcompositions may be formulated in liposomes such as, but not limited to,DiLa2 liposomes (Marina Biotech, Bothell, Wash.), SMARTICLES® (MarinaBiotech, Bothell, Wash.), neutral DOPC(1,2-dioleoyl-sn-glycero-3-phosphocholine) based liposomes (e.g., siRNAdelivery for ovarian cancer (Landen et al. Cancer Biology & Therapy 20065(12)1708-1713); herein incorporated by reference in its entirety) andhyaluronan-coated liposomes (Quiet Therapeutics, Israel).

In some embodiments, the cationic lipid may be a low molecular weightcationic lipid such as those described in U.S. Patent Application No.20130090372, the contents of which are herein incorporated by referencein their entirety.

In some embodiments, the RNA (e.g., mRNA) vaccines may be formulated ina lipid vesicle, which may have crosslinks between functionalized lipidbilayers.

In some embodiments, the RNA (e.g., mRNA) vaccines may be formulated ina lipid-polycation complex. The formation of the lipid-polycationcomplex may be accomplished by methods known in the art and/or asdescribed in U.S. Pub. No. 20120178702, herein incorporated by referencein its entirety. As a non-limiting example, the polycation may include acationic peptide or a polypeptide such as, but not limited to,polylysine, polyornithine and/or polyarginine. In some embodiments, theRNA (e.g., mRNA) vaccines may be formulated in a lipid-polycationcomplex, which may further include a non-cationic lipid such as, but notlimited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).

In some embodiments, the ratio of PEG in the lipid nanoparticle (LNP)formulations may be increased or decreased and/or the carbon chainlength of the PEG lipid may be modified from C14 to C18 to alter thepharmacokinetics and/or biodistribution of the LNP formulations. As anon-limiting example, LNP formulations may contain from about 0.5% toabout 3.0%, from about 1.0% to about 3.5%, from about 1.5% to about4.0%, from about 2.0% to about 4.5%, from about 2.5% to about 5.0%and/or from about 3.0% to about 6.0% of the lipid molar ratio ofPEG-c-DOMG(R-3-[ω-methoxy-poly(ethyleneglycol)2000)carbamoyl)]-1,2-dimyristyloxypropyl-3-amine)(also referred to herein as PEG-DOMG) as compared to the cationic lipid,DSPC and cholesterol. In some embodiments, the PEG-c-DOMG may bereplaced with a PEG lipid such as, but not limited to, PEG-DSG(1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG(1,2-Dimyristoyl-sn-glycerol) and/or PEG-DPG(1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The cationiclipid may be selected from any lipid known in the art such as, but notlimited to, DLin-MC3-DMA, DLin-DMA, C12-200 and DLin-KC2-DMA.

In some embodiments, the RNA (e.g., mRNA) vaccines may be formulated ina lipid nanoparticle.

In some embodiments, the RNA (e.g., mRNA) vaccine formulation comprisingthe polynucleotide is a nanoparticle which may comprise at least onelipid. The lipid may be selected from, but is not limited to, DLin-DMA,DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA,PEG, PEG-DMG, PEGylated lipids and amino alcohol lipids. In anotheraspect, the lipid may be a cationic lipid such as, but not limited to,DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA and aminoalcohol lipids. The amino alcohol cationic lipid may be the lipidsdescribed in and/or made by the methods described in U.S. PatentPublication No. US20130150625, herein incorporated by reference in itsentirety. As a non-limiting example, the cationic lipid may be2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol(Compound 1 in US20130150625);2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol(Compound 2 in US20130150625);2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol(Compound 3 in US20130150625); and2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol(Compound 4 in US20130150625); or any pharmaceutically acceptable saltor stereoisomer thereof.

Lipid nanoparticle formulations typically comprise a lipid, inparticular, an ionizable cationic lipid, for example,2,2-dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), ordi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), and furthercomprise a neutral lipid, a sterol and a molecule capable of reducingparticle aggregation, for example a PEG or PEG-modified lipid.

In some embodiments, the lipid nanoparticle formulation consistsessentially of (i) at least one lipid selected from the group consistingof 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); (ii) aneutral lipid selected from DSPC, DPPC, POPC, DOPE and SM; (iii) asterol, e.g., cholesterol; and (iv) a PEG-lipid, e.g., PEG-DMG orPEG-cDMA, in a molar ratio of about 20-60% cationic lipid:5-25% neutrallipid:25-55% sterol; 0.5-15% PEG-lipid.

In some embodiments, the formulation includes from about 25% to about75% on a molar basis of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g., fromabout 35 to about 65%, from about 45 to about 65%, about 60%, about57.5%, about 50% or about 40% on a molar basis.

In some embodiments, the formulation includes from about 0.5% to about15% on a molar basis of the neutral lipid e.g., from about 3 to about12%, from about 5 to about 10% or about 15%, about 10%, or about 7.5% ona molar basis. Examples of neutral lipids include, but are not limitedto, DSPC, POPC, DPPC, DOPE and SM. In some embodiments, the formulationincludes from about 5% to about 50% on a molar basis of the sterol(e.g., about 15 to about 45%, about 20 to about 40%, about 40%, about38.5%, about 35%, or about 31% on a molar basis. An exemplary sterol ischolesterol. In some embodiments, the formulation includes from about0.5% to about 20% on a molar basis of the PEG or PEG-modified lipid(e.g., about 0.5 to about 10%, about 0.5 to about 5%, about 1.5%, about0.5%, about 1.5%, about 3.5%, or about 5% on a molar basis). In someembodiments, the PEG or PEG modified lipid comprises a PEG molecule ofan average molecular weight of 2,000 Da. In other embodiments, the PEGor PEG modified lipid comprises a PEG molecule of an average molecularweight of less than 2,000, for example around 1,500 Da, around 1,000 Da,or around 500 Da. Examples of PEG-modified lipids include, but are notlimited to, PEG-distearoyl glycerol (PEG-DMG) (also referred herein asPEG-C14 or C14-PEG), PEG-cDMA (further discussed in Reyes et al. J.Controlled Release, 107, 276-287 (2005) the contents of which are hereinincorporated by reference in their entirety)

In some embodiments, the formulations of the present disclosure include25-75% of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 0.5-15% ofthe neutral lipid, 5-50% of the sterol, and 0.5-20% of the PEG orPEG-modified lipid on a molar basis.

In some embodiments, the formulations of the present disclosure include35-65% of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 3-12% of theneutral lipid, 15-45% of the sterol, and 0.5-10% of the PEG orPEG-modified lipid on a molar basis.

In some embodiments, the formulations of the present disclosure include45-65% of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 5-10% of theneutral lipid, 25-40% of the sterol, and 0.5-10% of the PEG orPEG-modified lipid on a molar basis.

In some embodiments, the formulations of the present disclosure includeabout 60% of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 7.5%of the neutral lipid, about 31% of the sterol, and about 1.5% of the PEGor PEG-modified lipid on a molar basis.

In some embodiments, the formulations of the present disclosure includeabout 50% of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 10% ofthe neutral lipid, about 38.5% of the sterol, and about 1.5% of the PEGor PEG-modified lipid on a molar basis.

In some embodiments, the formulations of the present disclosure includeabout 50% of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 10% ofthe neutral lipid, about 35% of the sterol, about 4.5% or about 5% ofthe PEG or PEG-modified lipid, and about 0.5% of the targeting lipid ona molar basis.

In some embodiments, the formulations of the present disclosure includeabout 40% of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 15% ofthe neutral lipid, about 40% of the sterol, and about 5% of the PEG orPEG-modified lipid on a molar basis.

In some embodiments, the formulations of the present disclosure includeabout 57.2% of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 7.1%of the neutral lipid, about 34.3% of the sterol, and about 1.4% of thePEG or PEG-modified lipid on a molar basis.

In some embodiments, the formulations of the present disclosure includeabout 57.5% of a cationic lipid selected from the PEG lipid is PEG-cDMA(PEG-cDMA is further discussed in Reyes et al. (J. Controlled Release,107, 276-287 (2005), the contents of which are herein incorporated byreference in their entirety), about 7.5% of the neutral lipid, about31.5% of the sterol, and about 3.5% of the PEG or PEG-modified lipid ona molar basis.

In some embodiments, lipid nanoparticle formulation consists essentiallyof a lipid mixture in molar ratios of about 20-70% cationic lipid:5-45%neutral lipid:20-55% cholesterol:0.5-15% PEG-modified lipid; morepreferably in a molar ratio of about 20-60% cationic lipid:5-25% neutrallipid:25-55% cholesterol:0.5-15% PEG-modified lipid.

In some embodiments, the molar lipid ratio is approximately50/10/38.5/1.5 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG, PEG-DSG or PEG-DPG),57.2/7.1134.3/1.4 (mol % cationic lipid/neutral lipid, e.g.,DPPC/Chol/PEG-modified lipid, e.g., PEG-cDMA), 40/15/40/5 (mol %cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g.,PEG-DMG), 50/10/35/4.5/0.5 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DSG), 50/10/35/5 (cationiclipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG),40/10/40/10 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA), 35/15/40/10(mol % cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid,e.g., PEG-DMG or PEG-cDMA) or 52/13/30/5 (mol % cationic lipid/neutrallipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA).

Examples of lipid nanoparticle compositions and methods of making sameare described, for example, in Semple et al. (2010) Nat. Biotechnol.28:172-176; Jayarama et al. (2012), Angew. Chem. Int. Ed., 51:8529-8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578 (thecontents of each of which are incorporated herein by reference in theirentirety).

In some embodiments, the LNP formulations of the RNA (e.g., mRNA)vaccines may contain PEG-c-DOMG at 3% lipid molar ratio. In someembodiments, the LNP formulations of the RNA (e.g., mRNA) vaccines maycontain PEG-c-DOMG at 1.5% lipid molar ratio.

In some embodiments, the pharmaceutical compositions of the RNA (e.g.,mRNA) vaccines may include at least one of the PEGylated lipidsdescribed in International Publication No. WO2012099755, the contents ofwhich are herein incorporated by reference in their entirety.

In some embodiments, the LNP formulation may contain PEG-DMG 2000(1,2-dimyristoyl-sn-glycero-3-phophoethanolamine-N-[methoxy(polyethyleneglycol)-2000). In some embodiments, the LNP formulation may containPEG-DMG 2000, a cationic lipid known in the art and at least one othercomponent. In some embodiments, the LNP formulation may contain PEG-DMG2000, a cationic lipid known in the art, DSPC and cholesterol. As anon-limiting example, the LNP formulation may contain PEG-DMG 2000,DLin-DMA, DSPC and cholesterol. As another non-limiting example the LNPformulation may contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol ina molar ratio of 2:40:10:48 (see e.g., Geall et al., Nonviral deliveryof self-amplifying RNA (e.g., mRNA) vaccines, PNAS 2012; PMID: 22908294,the contents of each of which are herein incorporated by reference intheir entirety).

The lipid nanoparticles described herein may be made in a sterileenvironment.

In some embodiments, the LNP formulation may be formulated in ananoparticle such as a nucleic acid-lipid particle. As a non-limitingexample, the lipid particle may comprise one or more active agents ortherapeutic agents; one or more cationic lipids comprising from about 50mol % to about 85 mol % of the total lipid present in the particle; oneor more non-cationic lipids comprising from about 13 mol % to about 49.5mol % of the total lipid present in the particle; and one or moreconjugated lipids that inhibit aggregation of particles comprising fromabout 0.5 mol % to about 2 mol % of the total lipid present in theparticle.

The nanoparticle formulations may comprise a phosphate conjugate. Thephosphate conjugate may increase in vivo circulation times and/orincrease the targeted delivery of the nanoparticle. As a non-limitingexample, the phosphate conjugates may include a compound of any one ofthe formulas described in International Application No. WO2013033438,the contents of which are herein incorporated by reference in itsentirety.

The nanoparticle formulation may comprise a polymer conjugate. Thepolymer conjugate may be a water-soluble conjugate. The polymerconjugate may have a structure as described in U.S. Patent ApplicationNo. 20130059360, the contents of which are herein incorporated byreference in its entirety. In some embodiments, polymer conjugates withthe polynucleotides of the present disclosure may be made using themethods and/or segmented polymeric reagents described in U.S. PatentApplication No. 20130072709, the contents of which are hereinincorporated by reference in its entirety. In some embodiments, thepolymer conjugate may have pendant side groups comprising ring moietiessuch as, but not limited to, the polymer conjugates described in U.S.Patent Publication No. US20130196948, the contents which are hereinincorporated by reference in its entirety.

The nanoparticle formulations may comprise a conjugate to enhance thedelivery of nanoparticles of the present disclosure in a subject.Further, the conjugate may inhibit phagocytic clearance of thenanoparticles in a subject. In one aspect, the conjugate may be a “self”peptide designed from the human membrane protein CD47 (e.g., the “self”particles described by Rodriguez et al. (Science 2013 339, 971-975),herein incorporated by reference in its entirety). As shown by Rodriguezet al., the self peptides delayed macrophage-mediated clearance ofnanoparticles which enhanced delivery of the nanoparticles. In anotheraspect, the conjugate may be the membrane protein CD47 (e.g., seeRodriguez et al. Science 2013 339, 971-975, herein incorporated byreference in its entirety). Rodriguez et al. showed that, similarly to“self” peptides, CD47 can increase the circulating particle ratio in asubject as compared to scrambled peptides and PEG coated nanoparticles.

In some embodiments, the RNA (e.g., mRNA) vaccines of the presentdisclosure are formulated in nanoparticles which comprise a conjugate toenhance the delivery of the nanoparticles of the present disclosure in asubject. The conjugate may be the CD47 membrane or the conjugate may bederived from the CD47 membrane protein, such as the “self” peptidedescribed previously. In some embodiments, the nanoparticle may comprisePEG and a conjugate of CD47 or a derivative thereof. In someembodiments, the nanoparticle may comprise both the “self” peptidedescribed above and the membrane protein CD47.

In some embodiments, a “self” peptide and/or CD47 protein may beconjugated to a virus-like particle or pseudovirion, as described hereinfor delivery of the RNA (e.g., mRNA) vaccines of the present disclosure.

In some embodiments, RNA (e.g., mRNA) vaccine pharmaceuticalcompositions comprising the polynucleotides of the present disclosureand a conjugate that may have a degradable linkage. Non-limitingexamples of conjugates include an aromatic moiety comprising anionizable hydrogen atom, a spacer moiety, and a water-soluble polymer.As a non-limiting example, pharmaceutical compositions comprising aconjugate with a degradable linkage and methods for delivering suchpharmaceutical compositions are described in U.S. Patent Publication No.US20130184443, the contents of which are herein incorporated byreference in their entirety.

The nanoparticle formulations may be a carbohydrate nanoparticlecomprising a carbohydrate carrier and a RNA (e.g., mRNA) vaccine. As anon-limiting example, the carbohydrate carrier may include, but is notlimited to, an anhydride-modified phytoglycogen or glycogen-typematerial, phytoglycogen octenyl succinate, phytoglycogen beta-dextrin,anhydride-modified phytoglycogen beta-dextrin. (See e.g., InternationalPublication No. WO2012109121; the contents of which are hereinincorporated by reference in their entirety).

Nanoparticle formulations of the present disclosure may be coated with asurfactant or polymer in order to improve the delivery of the particle.In some embodiments, the nanoparticle may be coated with a hydrophiliccoating such as, but not limited to, PEG coatings and/or coatings thathave a neutral surface charge. The hydrophilic coatings may help todeliver nanoparticles with larger payloads such as, but not limited to,RNA (e.g., mRNA) vaccines within the central nervous system. As anon-limiting example nanoparticles comprising a hydrophilic coating andmethods of making such nanoparticles are described in U.S. PatentPublication No. US20130183244, the contents of which are hereinincorporated by reference in their entirety.

In some embodiments, the lipid nanoparticles of the present disclosuremay be hydrophilic polymer particles. Non-limiting examples ofhydrophilic polymer particles and methods of making hydrophilic polymerparticles are described in U.S. Patent Publication No. US20130210991,the contents of which are herein incorporated by reference in theirentirety.

In some embodiments, the lipid nanoparticles of the present disclosuremay be hydrophobic polymer particles.

Lipid nanoparticle formulations may be improved by replacing thecationic lipid with a biodegradable cationic lipid which is known as arapidly eliminated lipid nanoparticle (reLNP). Ionizable cationiclipids, such as, but not limited to, DLinDMA, DLin-KC2-DMA, andDLin-MC3-DMA, have been shown to accumulate in plasma and tissues overtime and may be a potential source of toxicity. The rapid metabolism ofthe rapidly eliminated lipids can improve the tolerability andtherapeutic index of the lipid nanoparticles by an order of magnitudefrom a 1 mg/kg dose to a 10 mg/kg dose in rat. Inclusion of anenzymatically degraded ester linkage can improve the degradation andmetabolism profile of the cationic component, while still maintainingthe activity of the reLNP formulation. The ester linkage can beinternally located within the lipid chain or it may be terminallylocated at the terminal end of the lipid chain. The internal esterlinkage may replace any carbon in the lipid chain.

In some embodiments, the internal ester linkage may be located on eitherside of the saturated carbon.

In some embodiments, an immune response may be elicited by delivering alipid nanoparticle which may include a nanospecies, a polymer and animmunogen. (U.S. Publication No. 20120189700 and InternationalPublication No. WO2012099805; each of which is herein incorporated byreference in their entirety). The polymer may encapsulate thenanospecies or partially encapsulate the nanospecies. The immunogen maybe a recombinant protein, a modified RNA and/or a polynucleotidedescribed herein. In some embodiments, the lipid nanoparticle may beformulated for use in a vaccine such as, but not limited to, against apathogen.

Lipid nanoparticles may be engineered to alter the surface properties ofparticles so the lipid nanoparticles may penetrate the mucosal barrier.Mucus is located on mucosal tissue such as, but not limited to, oral(e.g., the buccal and esophageal membranes and tonsil tissue),ophthalmic, gastrointestinal (e.g., stomach, small intestine, largeintestine, colon, rectum), nasal, respiratory (e.g., nasal, pharyngeal,tracheal and bronchial membranes), genital (e.g., vaginal, cervical andurethral membranes). Nanoparticles larger than 10-200 nm which arepreferred for higher drug encapsulation efficiency and the ability toprovide the sustained delivery of a wide array of drugs have beenthought to be too large to rapidly diffuse through mucosal barriers.Mucus is continuously secreted, shed, discarded or digested and recycledso most of the trapped particles may be removed from the mucosa tissuewithin seconds or within a few hours. Large polymeric nanoparticles (200nm-500 nm in diameter) which have been coated densely with a lowmolecular weight polyethylene glycol (PEG) diffused through mucus only4- to 6-fold lower than the same particles diffusing in water (Lai etal. PNAS 2007 104:1482-487; Lai et al. Adv Drug Deliv Rev. 2009 61:158-171; each of which is herein incorporated by reference in itsentirety). The transport of nanoparticles may be determined using ratesof permeation and/or fluorescent microscopy techniques including, butnot limited to, fluorescence recovery after photobleaching (FRAP) andhigh resolution multiple particle tracking (MPT). As a non-limitingexample, compositions which can penetrate a mucosal barrier may be madeas described in U.S. Pat. No. 8,241,670 or International PatentPublication No. WO2013110028, the contents of each of which are hereinincorporated by reference in its entirety.

The lipid nanoparticle engineered to penetrate mucus may comprise apolymeric material (i.e. a polymeric core) and/or a polymer-vitaminconjugate and/or a tri-block co-polymer. The polymeric material mayinclude, but is not limited to, polyamines, polyethers, polyamides,polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes),polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes,polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates,polyacrylonitriles, and polyarylates. The polymeric material may bebiodegradable and/or biocompatible. Non-limiting examples ofbiocompatible polymers are described in International Patent PublicationNo. WO2013116804, the contents of which are herein incorporated byreference in their entirety. The polymeric material may additionally beirradiated. As a non-limiting example, the polymeric material may begamma irradiated (see e.g., International App. No. WO201282165, hereinincorporated by reference in its entirety). Non-limiting examples ofspecific polymers include poly(caprolactone) (PCL), ethylene vinylacetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid)(PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid)(PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide)(PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone),poly(D,L-lactide-co-caprolactone-co-glycolide),poly(D,L-lactide-co-PEO-co-D,L-lactide),poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate,polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA),polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids),polyanhydrides, polyorthoesters, poly(ester amides), polyamides,poly(ester ethers), polycarbonates, polyalkylenes such as polyethyleneand polypropylene, polyalkylene glycols such as poly(ethylene glycol)(PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such aspoly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinylethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halidessuch as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes,polystyrene (PS), polyurethanes, derivatized celluloses such as alkylcelluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters,nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose,polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA),poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate),poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate),poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate),poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) andcopolymers and mixtures thereof, polydioxanone and its copolymers,polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene,poloxamers, poly(ortho)esters, poly(butyric acid), poly(valeric acid),poly(lactide-co-caprolactone), PEG-PLGA-PEG and trimethylene carbonate,polyvinylpyrrolidone. The lipid nanoparticle may be coated or associatedwith a co-polymer such as, but not limited to, a block co-polymer (suchas a branched polyether-polyamide block copolymer described inInternational Publication No. WO2013012476, herein incorporated byreference in its entirety), and (poly(ethylene glycol))-(poly(propyleneoxide))-(poly(ethylene glycol)) triblock copolymer (see e.g., U.S.Publication 20120121718 and U.S. Publication 20100003337 and U.S. Pat.No. 8,263,665, the contents of each of which is herein incorporated byreference in their entirety). The co-polymer may be a polymer that isgenerally regarded as safe (GRAS) and the formation of the lipidnanoparticle may be in such a way that no new chemical entities arecreated. For example, the lipid nanoparticle may comprise poloxamerscoating PLGA nanoparticles without forming new chemical entities whichare still able to rapidly penetrate human mucus (Yang et al. Angew.Chem. Int. Ed. 2011 50:2597-2600; the contents of which are hereinincorporated by reference in their entirety). A non-limiting scalablemethod to produce nanoparticles which can penetrate human mucus isdescribed by Xu et al. (see, e.g., J Control Release 2013, 170:279-86;the contents of which are herein incorporated by reference in theirentirety).

The vitamin of the polymer-vitamin conjugate may be vitamin E. Thevitamin portion of the conjugate may be substituted with other suitablecomponents such as, but not limited to, vitamin A, vitamin E, othervitamins, cholesterol, a hydrophobic moiety, or a hydrophobic componentof other surfactants (e.g., sterol chains, fatty acids, hydrocarbonchains and alkylene oxide chains).

The lipid nanoparticle engineered to penetrate mucus may include surfacealtering agents such as, but not limited to, polynucleotides, anionicproteins (e.g., bovine serum albumin), surfactants (e.g., cationicsurfactants such as for example dimethyldioctadecyl-ammonium bromide),sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids,polymers (e.g., heparin, polyethylene glycol and poloxamer), mucolyticagents (e.g., N-acetylcysteine, mugwort, bromelain, papain,clerodendrum, acetylcysteine, bromhexine, carbocisteine, eprazinone,mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin,gelsolin, thymosin β4 dornase alfa, neltenexine, erdosteine) and variousDNases including rhDNase. The surface altering agent may be embedded orenmeshed in the particle's surface or disposed (e.g., by coating,adsorption, covalent linkage, or other process) on the surface of thelipid nanoparticle. (see e.g., U.S. Publication 20100215580 and U.S.Publication 20080166414 and US20130164343; the contents of each of whichare herein incorporated by reference in their entirety).

In some embodiments, the mucus penetrating lipid nanoparticles maycomprise at least one polynucleotide described herein. Thepolynucleotide may be encapsulated in the lipid nanoparticle and/ordisposed on the surface of the particle. The polynucleotide may becovalently coupled to the lipid nanoparticle. Formulations of mucuspenetrating lipid nanoparticles may comprise a plurality ofnanoparticles. Further, the formulations may contain particles which mayinteract with the mucus and alter the structural and/or adhesiveproperties of the surrounding mucus to decrease mucoadhesion, which mayincrease the delivery of the mucus penetrating lipid nanoparticles tothe mucosal tissue.

In some embodiments, the mucus penetrating lipid nanoparticles may be ahypotonic formulation comprising a mucosal penetration enhancingcoating. The formulation may be hypotonic for the epithelium to which itis being delivered. Non-limiting examples of hypotonic formulations maybe found in International Patent Publication No. WO2013110028, thecontents of which are herein incorporated by reference in theirentirety.

In some embodiments, in order to enhance the delivery through themucosal barrier the RNA (e.g., mRNA) vaccine formulation may comprise orbe a hypotonic solution. Hypotonic solutions were found to increase therate at which mucoinert particles such as, but not limited to,mucus-penetrating particles, were able to reach the vaginal epithelialsurface (see e.g., Ensign et al. Biomaterials 2013 34(28):6922-9, thecontents of which are herein incorporated by reference in theirentirety).

In some embodiments, the RNA (e.g., mRNA) vaccine is formulated as alipoplex, such as, without limitation, the ATUPLEX™® system, the DACCsystem, the DBTC system and other siRNA-lipoplex technology from SilenceTherapeutics (London, United Kingdom), STEMFECT™ from STEMGENT®(Cambridge, Mass.), and polyethylenimine (PEI) or protamine-basedtargeted and non-targeted delivery of nucleic acids (Aleku et al. CancerRes. 2008 68:9788-9798; Strumberg et al. Int J Clin Pharmacol Ther 201250:76-78; Santel et al., Gene Ther 2006 13:1222-1234; Santel et al.,Gene Ther 2006 13:1360-1370; Gutbier et al., Pulm Pharmacol. Ther. 201023:334-344; Kaufmann et al. Microvasc Res 2010 80:286-293 Weide et al. JImmunother. 2009 32:498-507; Weide et al. J Immunother. 2008 31:180-188;Pascolo Expert Opin. Biol. Ther. 4:1285-1294; Fotin-Mleczek et al., 2011J. Immunother. 34:1-15; Song et al., Nature Biotechnol. 2005,23:709-717; Peer et al., Proc Natl Acad Sci USA. 2007 6; 104:4095-4100;deFougerolles Hum Gene Ther. 2008 19:125-132, the contents of each ofwhich are incorporated herein by reference in their entirety).

In some embodiments, such formulations may also be constructed orcompositions altered such that they passively or actively are directedto different cell types in vivo, including but not limited tohepatocytes, immune cells, tumor cells, endothelial cells, antigenpresenting cells, and leukocytes (Akinc et al. Mol Ther. 201018:1357-1364; Song et al., Nat Biotechnol. 2005 23:709-717; Judge etal., J Clin Invest. 2009 119:661-673; Kaufmann et al., Microvasc Res2010 80:286-293; Santel et al., Gene Ther 2006 13:1222-1234; Santel etal., Gene Ther 2006 13:1360-1370; Gutbier et al., Pulm Pharmacol. Ther.2010 23:334-344; Basha et al., Mol. Ther. 2011 19:2186-2200; Fenske andCullis, Expert Opin Drug Deliv. 2008 5:25-44; Peer et al., Science. 2008319:627-630; Peer and Lieberman, Gene Ther. 2011 18:1127-1133, thecontents of each of which are incorporated herein by reference in theirentirety). One example of passive targeting of formulations to livercells includes the DLin-DMA, DLin-KC2-DMA and DLin-MC3-DMA-based lipidnanoparticle formulations, which have been shown to bind toapolipoprotein E and promote binding and uptake of these formulationsinto hepatocytes in vivo (Akinc et al. Mol Ther. 2010 18:1357-1364, thecontents of which are incorporated herein by reference in theirentirety). Formulations can also be selectively targeted throughexpression of different ligands on their surface as exemplified by, butnot limited by, folate, transferrin, N-acetylgalactosamine (GalNAc), andantibody targeted approaches (Kolhatkar et al., Curr Drug DiscovTechnol. 2011 8:197-206; Musacchio and Torchilin, Front Biosci. 201116:1388-1412; Yu et al., Mol Membr Biol. 2010 27:286-298; Patil et al.,Crit Rev Ther Drug Carrier Syst. 2008 25:1-61; Benoit et al.,Biomacromolecules. 2011 12:2708-2714; Zhao et al., Expert Opin DrugDeliv. 2008 5:309-319; Akinc et al., Mol Ther. 2010 18:1357-1364;Srinivasan et al., Methods Mol Biol. 2012 820:105-116; Ben-Arie et al.,Methods Mol Biol. 2012 757:497-507; Peer 2010 J Control Release.20:63-68; Peer et al., Proc Natl Acad Sci USA. 2007 104:4095-4100; Kimet al., Methods Mol Biol. 2011 721:339-353; Subramanya et al., Mol Ther.2010 18:2028-2037; Song et al., Nat Biotechnol. 2005 23:709-717; Peer etal., Science. 2008 319:627-630; Peer and Lieberman, Gene Ther. 201118:1127-1133, the contents of each of which are incorporated herein byreference in their entirety).

In some embodiments, the RNA (e.g., mRNA) vaccine is formulated as asolid lipid nanoparticle. A solid lipid nanoparticle (SLN) may bespherical with an average diameter between 10 to 1000 nm. SLNs possess asolid lipid core matrix that can solubilize lipophilic molecules and maybe stabilized with surfactants and/or emulsifiers. In some embodiments,the lipid nanoparticle may be a self-assembly lipid-polymer nanoparticle(see Zhang et al., ACS Nano, 2008, 2, pp 1696-1702; the contents ofwhich are herein incorporated by reference in their entirety). As anon-limiting example, the SLN may be the SLN described in InternationalPatent Publication No. WO2013105101, the contents of which are hereinincorporated by reference in their entirety. As another non-limitingexample, the SLN may be made by the methods or processes described inInternational Patent Publication No. WO2013105101, the contents of whichare herein incorporated by reference in their entirety.

Liposomes, lipoplexes, or lipid nanoparticles may be used to improve theefficacy of polynucleotides directed protein production as theseformulations may be able to increase cell transfection by the RNA (e.g.,mRNA) vaccine; and/or increase the translation of encoded protein. Onesuch example involves the use of lipid encapsulation to enable theeffective systemic delivery of polyplex plasmid DNA (Heyes et al., MolTher. 2007 15:713-720; the contents of which are incorporated herein byreference in their entirety). The liposomes, lipoplexes, or lipidnanoparticles may also be used to increase the stability of thepolynucleotide.

In some embodiments, the RNA (e.g., mRNA) vaccines of the presentdisclosure can be formulated for controlled release and/or targeteddelivery. As used herein, “controlled release” refers to apharmaceutical composition or compound release profile that conforms toa particular pattern of release to effect a therapeutic outcome. In someembodiments, the RNA (e.g., mRNA) vaccines may be encapsulated into adelivery agent described herein and/or known in the art for controlledrelease and/or targeted delivery. As used herein, the term “encapsulate”means to enclose, surround or encase. As it relates to the formulationof the compounds of the disclosure, encapsulation may be substantial,complete or partial. The term “substantially encapsulated” means that atleast greater than 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9,99.99 or greater than 99.999% of the pharmaceutical composition orcompound of the disclosure may be enclosed, surrounded or encased withinthe delivery agent. “Partially encapsulation” means that less than 10,10, 20, 30, 40, 50% or less of the pharmaceutical composition orcompound of the disclosure may be enclosed, surrounded or encased withinthe delivery agent. Advantageously, encapsulation may be determined bymeasuring the escape or the activity of the pharmaceutical compositionor compound of the disclosure using fluorescence and/or electronmicrograph. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80,85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.999% of thepharmaceutical composition or compound of the disclosure areencapsulated in the delivery agent.

In some embodiments, the controlled release formulation may include, butis not limited to, tri-block co-polymers. As a non-limiting example, theformulation may include two different types of tri-block co-polymers(International Pub. No. WO2012131104 and WO2012131106, the contents ofeach of which are incorporated herein by reference in their entirety).

In some embodiments, the RNA (e.g., mRNA) vaccines may be encapsulatedinto a lipid nanoparticle or a rapidly eliminated lipid nanoparticle andthe lipid nanoparticles or a rapidly eliminated lipid nanoparticle maythen be encapsulated into a polymer, hydrogel and/or surgical sealantdescribed herein and/or known in the art. As a non-limiting example, thepolymer, hydrogel or surgical sealant may be PLGA, ethylene vinylacetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua,Fla.), HYLENEX® (Halozyme Therapeutics, San Diego Calif.), surgicalsealants such as fibrinogen polymers (Ethicon Inc. Cornelia, Ga.),TISSELL® (Baxter International, Inc Deerfield, Ill.), PEG-basedsealants, and COSEAL® (Baxter International, Inc Deerfield, Ill.).

In some embodiments, the lipid nanoparticle may be encapsulated into anypolymer known in the art which may form a gel when injected into asubject. As another non-limiting example, the lipid nanoparticle may beencapsulated into a polymer matrix which may be biodegradable.

In some embodiments, the RNA (e.g., mRNA) vaccine formulation forcontrolled release and/or targeted delivery may also include at leastone controlled release coating. Controlled release coatings include, butare not limited to, OPADRY®, polyvinylpyrrolidone/vinyl acetatecopolymer, polyvinylpyrrolidone, hydroxypropyl methylcellulose,hydroxypropyl cellulose, hydroxyethyl cellulose, EUDRAGIT RL®, EUDRAGITRS® and cellulose derivatives such as ethylcellulose aqueous dispersions(AQUACOAT® and SURELEASE®).

In some embodiments, the RNA (e.g., mRNA) vaccine controlled releaseand/or targeted delivery formulation may comprise at least onedegradable polyester which may contain polycationic side chains.Degradeable polyesters include, but are not limited to, poly(serineester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester),and combinations thereof. In some embodiments, the degradable polyestersmay include a PEG conjugation to form a PEGylated polymer.

In some embodiments, the RNA (e.g., mRNA) vaccine controlled releaseand/or targeted delivery formulation comprising at least onepolynucleotide may comprise at least one PEG and/or PEG related polymerderivatives as described in U.S. Pat. No. 8,404,222, the contents ofwhich are incorporated herein by reference in their entirety.

In some embodiments, the RNA (e.g., mRNA) vaccine controlled releasedelivery formulation comprising at least one polynucleotide may be thecontrolled release polymer system described in US20130130348, thecontents of which are incorporated herein by reference in theirentirety.

In some embodiments, the RNA (e.g., mRNA) vaccines of the presentdisclosure may be encapsulated in a therapeutic nanoparticle, referredto herein as “therapeutic nanoparticle RNA (e.g., mRNA) vaccines.”Therapeutic nanoparticles may be formulated by methods described hereinand known in the art such as, but not limited to, International Pub Nos.WO2010005740, WO2010030763, WO2010005721, WO2010005723, WO2012054923,U.S. Publication Nos. US20110262491, US20100104645, US20100087337,US20100068285, US20110274759, US20100068286, US20120288541,US20130123351 and US20130230567 and U.S. Pat. Nos. 8,206,747, 8,293,276,8,318,208 and 8,318,211; the contents of each of which are hereinincorporated by reference in their entirety. In some embodiments,therapeutic polymer nanoparticles may be identified by the methodsdescribed in US Pub No. US20120140790, the contents of which are hereinincorporated by reference in their entirety.

In some embodiments, the therapeutic nanoparticle RNA (e.g., mRNA)vaccine may be formulated for sustained release. As used herein,“sustained release” refers to a pharmaceutical composition or compoundthat conforms to a release rate over a specific period of time. Theperiod of time may include, but is not limited to, hours, days, weeks,months and years. As a non-limiting example, the sustained releasenanoparticle may comprise a polymer and a therapeutic agent such as, butnot limited to, the polynucleotides of the present disclosure (seeInternational Pub No. 2010075072 and US Pub No. US20100216804,US20110217377 and US20120201859, the contents of each of which areincorporated herein by reference in their entirety). In anothernon-limiting example, the sustained release formulation may compriseagents which permit persistent bioavailability such as, but not limitedto, crystals, macromolecular gels and/or particulate suspensions (seeU.S. Patent Publication No. US20130150295, the contents of each of whichare incorporated herein by reference in their entirety).

In some embodiments, the therapeutic nanoparticle RNA (e.g., mRNA)vaccines may be formulated to be target specific. As a non-limitingexample, the therapeutic nanoparticles may include a corticosteroid (seeInternational Pub. No. WO2011084518, the contents of which areincorporated herein by reference in their entirety). As a non-limitingexample, the therapeutic nanoparticles may be formulated innanoparticles described in International Pub No. WO2008121949,WO2010005726, WO2010005725, WO2011084521 and US Pub No. US20100069426,US20120004293 and US20100104655, the contents of each of which areincorporated herein by reference in their entirety.

In some embodiments, the nanoparticles of the present disclosure maycomprise a polymeric matrix. As a non-limiting example, the nanoparticlemay comprise two or more polymers such as, but not limited to,polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids,polypropylfumerates, polycaprolactones, polyamides, polyacetals,polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinylalcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polycyanoacrylates, polyureas, polystyrenes,polyamines, polylysine, poly(ethylene imine), poly(serine ester),poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) orcombinations thereof.

In some embodiments, the therapeutic nanoparticle comprises a diblockcopolymer. In some embodiments, the diblock copolymer may include PEG incombination with a polymer such as, but not limited to, polyethylenes,polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates,polycaprolactones, polyamides, polyacetals, polyethers, polyesters,poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine,poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine),poly(4-hydroxy-L-proline ester) or combinations thereof. In yet anotherembodiment, the diblock copolymer may be a high-X diblock copolymer suchas those described in International Patent Publication No. WO2013120052,the contents of which are incorporated herein by reference in theirentirety.

As a non-limiting example the therapeutic nanoparticle comprises aPLGA-PEG block copolymer (see U.S. Publication No. US20120004293 andU.S. Pat. No. 8,236,330, each of which is herein incorporated byreference in their entirety). In another non-limiting example, thetherapeutic nanoparticle is a stealth nanoparticle comprising a diblockcopolymer of PEG and PLA or PEG and PLGA (see U.S. Pat. No. 8,246,968and International Publication No. WO2012166923, the contents of each ofwhich are herein incorporated by reference in their entirety). In yetanother non-limiting example, the therapeutic nanoparticle is a stealthnanoparticle or a target-specific stealth nanoparticle as described inU.S. Patent Publication No. US20130172406, the contents of which areherein incorporated by reference in their entirety.

In some embodiments, the therapeutic nanoparticle may comprise amultiblock copolymer (see e.g., U.S. Pat. Nos. 8,263,665 and 8,287,910and U.S. Patent Pub. No. US20130195987, the contents of each of whichare herein incorporated by reference in their entirety).

In yet another non-limiting example, the lipid nanoparticle comprisesthe block copolymer PEG-PLGA-PEG (see e.g., the thermosensitive hydrogel(PEG-PLGA-PEG) was used as a TGF-beta1 gene delivery vehicle in Lee etal. Thermosensitive Hydrogel as a TGF-β1 Gene Delivery Vehicle EnhancesDiabetic Wound Healing. Pharmaceutical Research, 2003 20(12): 1995-2000;as a controlled gene delivery system in Li et al. Controlled GeneDelivery System Based on Thermosensitive Biodegradable Hydrogel.Pharmaceutical Research 2003 20:884-888; and Chang et al., Non-ionicamphiphilic biodegradable PEG-PLGA-PEG copolymer enhances gene deliveryefficiency in rat skeletal muscle. J Controlled Release. 2007118:245-253, the contents of each of which are herein incorporated byreference in their entirety). The RNA (e.g., mRNA) vaccines of thepresent disclosure may be formulated in lipid nanoparticles comprisingthe PEG-PLGA-PEG block copolymer.

In some embodiments, the therapeutic nanoparticle may comprise amultiblock copolymer (see e.g., U.S. Pat. Nos. 8,263,665 and 8,287,910and U.S. Patent Pub. No. US20130195987, the contents of each of whichare herein incorporated by reference in their entirety).

In some embodiments, the block copolymers described herein may beincluded in a polyion complex comprising a non-polymeric micelle and theblock copolymer. (see e.g., U.S. Publication No. 20120076836, thecontents of which are herein incorporated by reference in theirentirety).

In some embodiments, the therapeutic nanoparticle may comprise at leastone acrylic polymer. Acrylic polymers include but are not limited to,acrylic acid, methacrylic acid, acrylic acid and methacrylic acidcopolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates,cyanoethyl methacrylate, amino alkyl methacrylate copolymer,poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates andcombinations thereof.

In some embodiments, the therapeutic nanoparticles may comprise at leastone poly(vinyl ester) polymer. The poly(vinyl ester) polymer may be acopolymer such as a random copolymer. As a non-limiting example, therandom copolymer may have a structure such as those described inInternational Application No. WO2013032829 or U.S. Patent Publication NoUS20130121954, the contents of each of which are herein incorporated byreference in their entirety. In some embodiments, the poly(vinyl ester)polymers may be conjugated to the polynucleotides described herein.

In some embodiments, the therapeutic nanoparticle may comprise at leastone diblock copolymer. The diblock copolymer may be, but it not limitedto, a poly(lactic) acid-poly(ethylene)glycol copolymer (see, e.g.,International Patent Publication No. WO2013044219, the contents of whichare herein incorporated by reference in their entirety). As anon-limiting example, the therapeutic nanoparticle may be used to treatcancer (see International publication No. WO2013044219, the contents ofwhich are herein incorporated by reference in their entirety).

In some embodiments, the therapeutic nanoparticles may comprise at leastone cationic polymer described herein and/or known in the art.

In some embodiments, the therapeutic nanoparticles may comprise at leastone amine-containing polymer such as, but not limited to polylysine,polyethylene imine, poly(amidoamine) dendrimers, poly(beta-amino esters)(see, e.g., U.S. Pat. No. 8,287,849, the contents of which are hereinincorporated by reference in their entirety) and combinations thereof.

In some embodiments, the nanoparticles described herein may comprise anamine cationic lipid such as those described in International PatentApplication No. WO2013059496, the contents of which are hereinincorporated by reference in their entirety. In some embodiments, thecationic lipids may have an amino-amine or an amino-amide moiety.

In some embodiments, the therapeutic nanoparticles may comprise at leastone degradable polyester which may contain polycationic side chains.Degradeable polyesters include, but are not limited to, poly(serineester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester),and combinations thereof. In some embodiments, the degradable polyestersmay include a PEG conjugation to form a PEGylated polymer.

In some embodiments, the synthetic nanocarriers may contain animmunostimulatory agent to enhance the immune response from delivery ofthe synthetic nanocarrier. As a non-limiting example, the syntheticnanocarrier may comprise a Th1 immunostimulatory agent, which mayenhance a Th1-based response of the immune system (see International PubNo. WO2010123569 and U.S. Publication No. US20110223201, the contents ofeach of which are herein incorporated by reference in their entirety).

In some embodiments, the synthetic nanocarriers may be formulated fortargeted release. In some embodiments, the synthetic nanocarrier isformulated to release the polynucleotides at a specified pH and/or aftera desired time interval. As a non-limiting example, the syntheticnanoparticle may be formulated to release the RNA (e.g., mRNA) vaccinesafter 24 hours and/or at a pH of 4.5 (see International Publication Nos.WO2010138193 and WO2010138194 and US Pub Nos. US20110020388 andUS20110027217, each of which is herein incorporated by reference intheir entireties).

In some embodiments, the synthetic nanocarriers may be formulated forcontrolled and/or sustained release of the polynucleotides describedherein. As a non-limiting example, the synthetic nanocarriers forsustained release may be formulated by methods known in the art,described herein and/or as described in International Pub No.WO2010138192 and US Pub No. 20100303850, each of which is hereinincorporated by reference in their entirety.

In some embodiments, the RNA (e.g., mRNA) vaccine may be formulated forcontrolled and/or sustained release wherein the formulation comprises atleast one polymer that is a crystalline side chain (CYSC) polymer. CYSCpolymers are described in U.S. Pat. No. 8,399,007, herein incorporatedby reference in its entirety.

In some embodiments, the synthetic nanocarrier may be formulated for useas a vaccine. In some embodiments, the synthetic nanocarrier mayencapsulate at least one polynucleotide which encode at least oneantigen. As a non-limiting example, the synthetic nanocarrier mayinclude at least one antigen and an excipient for a vaccine dosage form(see International Publication No. WO2011150264 and U.S. Publication No.US20110293723, the contents of each of which are herein incorporated byreference in their entirety). As another non-limiting example, a vaccinedosage form may include at least two synthetic nanocarriers with thesame or different antigens and an excipient (see InternationalPublication No. WO2011150249 and U.S. Publication No. US20110293701, thecontents of each of which are herein incorporated by reference in theirentirety). The vaccine dosage form may be selected by methods describedherein, known in the art and/or described in International PublicationNo. WO2011150258 and U.S. Publication No. US20120027806, the contents ofeach of which are herein incorporated by reference in their entirety).

In some embodiments, the synthetic nanocarrier may comprise at least onepolynucleotide which encodes at least one adjuvant. As non-limitingexample, the adjuvant may comprise dimethyldioctadecylammonium-bromide,dimethyldioctadecylammonium-chloride,dimethyldioctadecylammonium-phosphate ordimethyldioctadecylammonium-acetate (DDA) and an apolar fraction or partof said apolar fraction of a total lipid extract of a mycobacterium(see, e.g., U.S. Pat. No. 8,241,610, the content of which is hereinincorporated by reference in its entirety). In some embodiments, thesynthetic nanocarrier may comprise at least one polynucleotide and anadjuvant. As a non-limiting example, the synthetic nanocarriercomprising and adjuvant may be formulated by the methods described inInternational Publication No. WO2011150240 and U.S. Publication No.US20110293700, the contents of each of which are herein incorporated byreference in their entirety.

In some embodiments, the synthetic nanocarrier may encapsulate at leastone polynucleotide that encodes a peptide, fragment or region from avirus. As a non-limiting example, the synthetic nanocarrier may include,but is not limited to, any of the nanocarriers described inInternational Publication No. WO2012024621, WO201202629, WO2012024632and U.S. Publication No. US20120064110, US20120058153 and US20120058154,the contents of each of which are herein incorporated by reference intheir entirety.

In some embodiments, the synthetic nanocarrier may be coupled to apolynucleotide which may be able to trigger a humoral and/or cytotoxic Tlymphocyte (CTL) response (see, e.g., International Publication No.WO2013019669, the contents of which are herein incorporated by referencein their entirety).

In some embodiments, the RNA (e.g., mRNA) vaccine may be encapsulatedin, linked to and/or associated with zwitterionic lipids. Non-limitingexamples of zwitterionic lipids and methods of using zwitterionic lipidsare described in U.S. Patent Publication No. US20130216607, the contentsof which are herein incorporated by reference in their entirety. In someaspects, the zwitterionic lipids may be used in the liposomes and lipidnanoparticles described herein.

In some embodiments, the RNA (e.g., mRNA) vaccine may be formulated incolloid nanocarriers as described in U.S. Patent Publication No.US20130197100, the contents of which are herein incorporated byreference in their entirety.

In some embodiments, the nanoparticle may be optimized for oraladministration. The nanoparticle may comprise at least one cationicbiopolymer such as, but not limited to, chitosan or a derivativethereof. As a non-limiting example, the nanoparticle may be formulatedby the methods described in U.S. Publication No. 20120282343, thecontents of which are herein incorporated by reference in theirentirety.

In some embodiments, LNPs comprise the lipid KL52 (an amino-lipiddisclosed in U.S. Application Publication No. 2012/0295832, the contentsof which are herein incorporated by reference in their entirety.Activity and/or safety (as measured by examining one or more of ALT/AST,white blood cell count and cytokine induction, for example) of LNPadministration may be improved by incorporation of such lipids. LNPscomprising KL52 may be administered intravenously and/or in one or moredoses. In some embodiments, administration of LNPs comprising KL52results in equal or improved mRNA and/or protein expression as comparedto LNPs comprising MC3.

In some embodiments, RNA (e.g., mRNA) vaccine may be delivered usingsmaller LNPs. Such particles may comprise a diameter from below 0.1 umup to 100 nm such as, but not limited to, less than 0.1 um, less than1.0 um, less than 5 um, less than 10 um, less than 15 um, less than 20um, less than 25 um, less than 30 um, less than 35 um, less than 40 um,less than 50 um, less than 55 um, less than 60 um, less than 65 um, lessthan 70 um, less than 75 um, less than 80 um, less than 85 um, less than90 um, less than 95 um, less than 100 um, less than 125 um, less than150 um, less than 175 um, less than 200 um, less than 225 um, less than250 um, less than 275 um, less than 300 um, less than 325 um, less than350 um, less than 375 um, less than 400 um, less than 425 um, less than450 um, less than 475 um, less than 500 um, less than 525 um, less than550 um, less than 575 um, less than 600 um, less than 625 um, less than650 um, less than 675 um, less than 700 um, less than 725 um, less than750 um, less than 775 um, less than 800 um, less than 825 um, less than850 um, less than 875 um, less than 900 um, less than 925 um, less than950 um, less than 975 um, or less than 1000 um.

In some embodiments, RNA (e.g., mRNA) vaccines may be delivered usingsmaller LNPs, which may comprise a diameter from about 1 nm to about 100nm, from about 1 nm to about 10 nm, about 1 nm to about 20 nm, fromabout 1 nm to about 30 nm, from about 1 nm to about 40 nm, from about 1nm to about 50 nm, from about 1 nm to about 60 nm, from about 1 nm toabout 70 nm, from about 1 nm to about 80 nm, from about 1 nm to about 90nm, from about 5 nm to about from 100 nm, from about 5 nm to about 10nm, about 5 nm to about 20 nm, from about 5 nm to about 30 nm, fromabout 5 nm to about 40 nm, from about 5 nm to about 50 nm, from about 5nm to about 60 nm, from about 5 nm to about 70 nm, from about 5 nm toabout 80 nm, from about 5 nm to about 90 nm, about 10 to about 50 nm,from about 20 to about 50 nm, from about 30 to about 50 nm, from about40 to about 50 nm, from about 20 to about 60 nm, from about 30 to about60 nm, from about 40 to about 60 nm, from about 20 to about 70 nm, fromabout 30 to about 70 nm, from about 40 to about 70 nm, from about 50 toabout 70 nm, from about 60 to about 70 nm, from about 20 to about 80 nm,from about 30 to about 80 nm, from about 40 to about 80 nm, from about50 to about 80 nm, from about 60 to about 80 nm, from about 20 to about90 nm, from about 30 to about 90 nm, from about 40 to about 90 nm, fromabout 50 to about 90 nm, from about 60 to about 90 nm and/or from about70 to about 90 nm.

In some embodiments, such LNPs are synthesized using methods comprisingmicrofluidic mixers. Examples of microfluidic mixers may include, butare not limited to, a slit interdigital micromixer including, but notlimited to those manufactured by Microinnova (Allerheiligen bei Wildon,Austria) and/or a staggered herringbone micromixer (SHM) (Zhigaltsev, I.V. et al., Bottom-up design and synthesis of limit size lipidnanoparticle systems with aqueous and triglyceride cores usingmillisecond microfluidic mixing have been published (Langmuir. 2012.28:3633-40; Belliveau, N. M. et al., Microfluidic synthesis of highlypotent limit-size lipid nanoparticles for in vivo delivery of siRNA.Molecular Therapy-Nucleic Acids. 2012. 1:e37; Chen, D. et al., Rapiddiscovery of potent siRNA-containing lipid nanoparticles enabled bycontrolled microfluidic formulation. J Am Chem Soc. 2012.134(16):6948-51, the contents of each of which are herein incorporatedby reference in their entirety). In some embodiments, methods of LNPgeneration comprising SHM, further comprise the mixing of at least twoinput streams wherein mixing occurs by microstructure-induced chaoticadvection (MICA). According to this method, fluid streams flow throughchannels present in a herringbone pattern, causing rotational flow andfolding the fluids around each other. This method may also comprise asurface for fluid mixing wherein the surface changes orientations duringfluid cycling. Methods of generating LNPs using SHM include thosedisclosed in U.S. Application Publication Nos. 2004/0262223 and2012/0276209, the contents of each of which are herein incorporated byreference in their entirety.

In some embodiments, the RNA (e.g., mRNA) vaccine of the presentdisclosure may be formulated in lipid nanoparticles created using amicromixer such as, but not limited to, a Slit InterdigitalMicrostructured Mixer (SIMM-V2) or a Standard Slit Interdigital MicroMixer (SSIMM) or Caterpillar (CPMM) or Impinging-jet (IJMM) from theInstitut für Mikrotechnik Mainz GmbH, Mainz Germany.

In some embodiments, the RNA (e.g., mRNA) vaccines of the presentdisclosure may be formulated in lipid nanoparticles created usingmicrofluidic technology (see, e.g., Whitesides, George M. The Originsand the Future of Microfluidics. Nature, 2006 442: 368-373; and Abrahamet al. Chaotic Mixer for Microchannels. Science, 2002 295: 647-651; eachof which is herein incorporated by reference in its entirety). As anon-limiting example, controlled microfluidic formulation includes apassive method for mixing streams of steady pressure-driven flows inmicro channels at a low Reynolds number (see, e.g., Abraham et al.Chaotic Mixer for Microchannels. Science, 2002 295: 647-651, thecontents of which are herein incorporated by reference in theirentirety).

In some embodiments, the RNA (e.g., mRNA) vaccines of the presentdisclosure may be formulated in lipid nanoparticles created using amicromixer chip such as, but not limited to, those from HarvardApparatus (Holliston, Mass.) or Dolomite Microfluidics (Royston, UK). Amicromixer chip can be used for rapid mixing of two or more fluidstreams with a split and recombine mechanism.

In some embodiments, the RNA (e.g., mRNA) vaccines of the disclosure maybe formulated for delivery using the drug encapsulating microspheresdescribed in International Patent Publication No. WO2013063468 or U.S.Pat. No. 8,440,614, the contents of each of which are hereinincorporated by reference in their entirety. The microspheres maycomprise a compound of the formula (I), (II), (III), (IV), (V) or (VI)as described in International Patent Publication No. WO2013063468, thecontents of which are herein incorporated by reference in theirentirety. In some embodiments, the amino acid, peptide, polypeptide,and/or lipids are useful in delivering the RNA (e.g., mRNA) vaccines ofthe disclosure to cells (see International Patent Publication No.WO2013063468, the contents of which are herein incorporated by referencein their entirety).

In some embodiments, the RNA (e.g., mRNA) vaccines of the disclosure maybe formulated in lipid nanoparticles having a diameter from about 10 toabout 100 nm such as, but not limited to, about 10 to about 20 nm, about10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm,about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 toabout 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm,about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50 toabout 60 nm, about 50 to about 70 nm about 50 to about 80 nm, about 50to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm,about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100nm, about 80 to about 90 nm, about 80 to about 100 nm and/or about 90 toabout 100 nm.

In some embodiments, the lipid nanoparticles may have a diameter fromabout 10 to 500 nm.

In some embodiments, the lipid nanoparticle may have a diameter greaterthan 100 nm, greater than 150 nm, greater than 200 nm, greater than 250nm, greater than 300 nm, greater than 350 nm, greater than 400 nm,greater than 450 nm, greater than 500 nm, greater than 550 nm, greaterthan 600 nm, greater than 650 nm, greater than 700 nm, greater than 750nm, greater than 800 nm, greater than 850 nm, greater than 900 nm,greater than 950 nm or greater than 1000 nm.

In some embodiments, the lipid nanoparticle may be a limit size lipidnanoparticle described in International Patent Publication No.WO2013059922, the contents of which are herein incorporated by referencein their entirety. The limit size lipid nanoparticle may comprise alipid bilayer surrounding an aqueous core or a hydrophobic core; wherethe lipid bilayer may comprise a phospholipid such as, but not limitedto, diacylphosphatidylcholine, a diacylphosphatidylethanolamine, aceramide, a sphingomyelin, a dihydrosphingomyelin, a cephalin, acerebroside, a C8-C20 fatty acid diacylphophatidylcholine, and1-palmitoyl-2-oleoyl phosphatidylcholine (POPC). In some embodiments,the limit size lipid nanoparticle may comprise a polyethyleneglycol-lipid such as, but not limited to, DLPE-PEG, DMPE-PEG, DPPC-PEGand DSPE-PEG.

In some embodiments, the RNA (e.g., mRNA) vaccines may be delivered,localized and/or concentrated in a specific location using the deliverymethods described in International Patent Publication No. WO2013063530,the contents of which are herein incorporated by reference in theirentirety. As a non-limiting example, a subject may be administered anempty polymeric particle prior to, simultaneously with or afterdelivering the RNA (e.g., mRNA) vaccines to the subject. The emptypolymeric particle undergoes a change in volume once in contact with thesubject and becomes lodged, embedded, immobilized or entrapped at aspecific location in the subject.

In some embodiments, the RNA (e.g., mRNA) vaccines may be formulated inan active substance release system (see, e.g., U.S. Patent PublicationNo. US20130102545, the contents of which are herein incorporated byreference in their entirety). The active substance release system maycomprise 1) at least one nanoparticle bonded to an oligonucleotideinhibitor strand which is hybridized with a catalytically active nucleicacid and 2) a compound bonded to at least one substrate molecule bondedto a therapeutically active substance (e.g., polynucleotides describedherein), where the therapeutically active substance is released by thecleavage of the substrate molecule by the catalytically active nucleicacid.

In some embodiments, the RNA (e.g., mRNA) vaccines may be formulated ina nanoparticle comprising an inner core comprising a non-cellularmaterial and an outer surface comprising a cellular membrane. Thecellular membrane may be derived from a cell or a membrane derived froma virus. As a non-limiting example, the nanoparticle may be made by themethods described in International Patent Publication No. WO2013052167,the contents of which are herein incorporated by reference in theirentirety. As another non-limiting example, the nanoparticle described inInternational Patent Publication No. WO2013052167, the contents of whichare herein incorporated by reference in their entirety, may be used todeliver the RNA (e.g., mRNA) vaccines described herein.

In some embodiments, the RNA (e.g., mRNA) vaccines may be formulated inporous nanoparticle-supported lipid bilayers (protocells). Protocellsare described in International Patent Publication No. WO2013056132, thecontents of which are herein incorporated by reference in theirentirety.

In some embodiments, the RNA (e.g., mRNA) vaccines described herein maybe formulated in polymeric nanoparticles as described in or made by themethods described in U.S. Pat. Nos. 8,420,123 and 8,518,963 and EuropeanPatent No. EP2073848B1, the contents of each of which are hereinincorporated by reference in their entirety. As a non-limiting example,the polymeric nanoparticle may have a high glass transition temperaturesuch as the nanoparticles described in or nanoparticles made by themethods described in U.S. Pat. No. 8,518,963, the contents of which areherein incorporated by reference in their entirety. As anothernon-limiting example, the polymer nanoparticle for oral and parenteralformulations may be made by the methods described in European Patent No.EP2073848B1, the contents of which are herein incorporated by referencein their entirety.

In some embodiments, the RNA (e.g., mRNA) vaccines described herein maybe formulated in nanoparticles used in imaging. The nanoparticles may beliposome nanoparticles such as those described in U.S. PatentPublication No US20130129636, herein incorporated by reference in itsentirety. As a non-limiting example, the liposome may comprisegadolinium(III)2-{4,7-bis-carboxymethyl-10-[(N,N-distearylamidomethyl-N′-amido-methyl]-1,4,7,10-tetra-azacyclododec-1-yl}-aceticacid and a neutral, fully saturated phospholipid component (see, e.g.,U.S. Patent Publication No US20130129636, the contents of which areherein incorporated by reference in their entirety).

In some embodiments, the nanoparticles which may be used in the presentdisclosure are formed by the methods described in U.S. PatentApplication No. US20130130348, the contents of which are hereinincorporated by reference in their entirety.

The nanoparticles of the present disclosure may further includenutrients such as, but not limited to, those which deficiencies can leadto health hazards from anemia to neural tube defects (see, e.g., thenanoparticles described in International Patent Publication NoWO2013072929, the contents of which are herein incorporated by referencein their entirety). As a non-limiting example, the nutrient may be ironin the form of ferrous, ferric salts or elemental iron, iodine, folicacid, vitamins or micronutrients.

In some embodiments, the RNA (e.g., mRNA) vaccines of the presentdisclosure may be formulated in a swellable nanoparticle. The swellablenanoparticle may be, but is not limited to, those described in U.S. Pat.No. 8,440,231, the contents of which are herein incorporated byreference in their entirety. As a non-limiting embodiment, the swellablenanoparticle may be used for delivery of the RNA (e.g., mRNA) vaccinesof the present disclosure to the pulmonary system (see, e.g., U.S. Pat.No. 8,440,231, the contents of which are herein incorporated byreference in their entirety).

The RNA (e.g., mRNA) vaccines of the present disclosure may beformulated in polyanhydride nanoparticles such as, but not limited to,those described in U.S. Pat. No. 8,449,916, the contents of which areherein incorporated by reference in their entirety.

The nanoparticles and microparticles of the present disclosure may begeometrically engineered to modulate macrophage and/or the immuneresponse. In some embodiments, the geometrically engineered particlesmay have varied shapes, sizes and/or surface charges in order toincorporated the polynucleotides of the present disclosure for targeteddelivery such as, but not limited to, pulmonary delivery (see, e.g.,International Publication No WO2013082111, the contents of which areherein incorporated by reference in their entirety). Other physicalfeatures the geometrically engineering particles may have include, butare not limited to, fenestrations, angled arms, asymmetry and surfaceroughness, and charge which can alter the interactions with cells andtissues. As a non-limiting example, nanoparticles of the presentdisclosure may be made by the methods described in InternationalPublication No WO2013082111, the contents of which are hereinincorporated by reference in their entirety.

In some embodiments, the nanoparticles of the present disclosure may bewater soluble nanoparticles such as, but not limited to, those describedin International Publication No. WO2013090601, the contents of which areherein incorporated by reference in their entirety. The nanoparticlesmay be inorganic nanoparticles which have a compact and zwitterionicligand in order to exhibit good water solubility. The nanoparticles mayalso have small hydrodynamic diameters (HD), stability with respect totime, pH, and salinity and a low level of non-specific protein binding.

In some embodiments the nanoparticles of the present disclosure may bedeveloped by the methods described in U.S. Patent Publication No.US20130172406, the contents of which are herein incorporated byreference in their entirety.

In some embodiments, the nanoparticles of the present disclosure arestealth nanoparticles or target-specific stealth nanoparticles such as,but not limited to, those described in U.S. Patent Publication No.US20130172406, the contents of which are herein incorporated byreference in their entirety. The nanoparticles of the present disclosuremay be made by the methods described in U.S. Patent Publication No.US20130172406, the contents of which are herein incorporated byreference in their entirety.

In some embodiments, the stealth or target-specific stealthnanoparticles may comprise a polymeric matrix. The polymeric matrix maycomprise two or more polymers such as, but not limited to,polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids,polypropylfumerates, polycaprolactones, polyamides, polyacetals,polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinylalcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polycyanoacrylates, polyureas, polystyrenes,polyamines, polyesters, polyanhydrides, polyethers, polyurethanes,polymethacrylates, polyacrylates, polycyanoacrylates or combinationsthereof.

In some embodiments, the nanoparticle may be a nanoparticle-nucleic acidhybrid structure having a high density nucleic acid layer. As anon-limiting example, the nanoparticle-nucleic acid hybrid structure maymade by the methods described in U.S. Patent Publication No.US20130171646, the contents of which are herein incorporated byreference in their entirety. The nanoparticle may comprise a nucleicacid such as, but not limited to, polynucleotides described hereinand/or known in the art.

At least one of the nanoparticles of the present disclosure may beembedded in the core of a nanostructure or coated with a low densityporous 3-D structure or coating which is capable of carrying orassociating with at least one payload within or on the surface of thenanostructure. Non-limiting examples of the nanostructures comprising atleast one nanoparticle are described in International Patent PublicationNo. WO2013123523, the contents of which are herein incorporated byreference in their entirety.

In some embodiments the RNA (e.g., mRNA) vaccine may be associated witha cationic or polycationic compounds, including protamine, nucleoline,spermine or spermidine, or other cationic peptides or proteins, such aspoly-L-lysine (PLL), polyarginine, basic polypeptides, cell penetratingpeptides (CPPs), including HIV-binding peptides, HIV-1 Tat (HIV),Tat-derived peptides, Penetratin, VP²² derived or analog peptides,Pestivirus Ems, HSINV, VP²² (Herpes simplex), MAP, KALA or proteintransduction domains (PTDs), PpT620, prolin-rich peptides, arginine-richpeptides, lysine-rich peptides, MPG-peptide(s), Pep-1, L-oligomers,Calcitonin peptide(s), Antennapedia-derived peptides (particularly fromDrosophila antennapedia), pAntp, plsl, FGF, Lactoferrin, Transportan,Buforin-2, Bac715-24, SynB, SynB, pVEC, hCT-derived peptides, SAP,histones, cationic polysaccharides, for example chitosan, polybrene,cationic polymers, e.g. polyethyleneimine (PEI), cationic lipids, e.g.DOTMA: [1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride,DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP,DOPE: Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC,DOGS: Dioctadecylamidoglicylspermin, DIMRI: Dimyristooxypropyl dimethylhydroxyethyl ammonium bromide, DOTAP:dioleoyloxy-3-(trimethylammonio)propane, DC-6-14:O,O-ditetradecanoyl-N-.alpha.-trimethylammonioacetyl)diethanolaminechloride, CLIP 1:rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammoniumchloride, CLIP6:rac-[2(2,3-dihexadecyloxypropyloxymethyloxy)ethyl]-trimethylammonium,CLIP9:rac-[2(2,3-dihexadecyloxypropyloxysuccinyloxy)ethyl]-trimethylammo-nium,oligofectamine, or cationic or polycationic polymers, e.g. modifiedpolyaminoacids, such as beta-aminoacid-polymers or reversed polyamides,etc., modified polyethylenes, such as PVP(poly(N-ethyl-4-vinylpyridinium bromide)), etc., modified acrylates,such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc.,modified amidoamines such as pAMAM (poly(amidoamine)), etc., modifiedpolybetaminoester (PBAE), such as diamine end modified 1,4 butanedioldiacrylate-co-5-amino-1-pentanol polymers, etc., dendrimers, such aspolypropylamine dendrimers or pAMAM based dendrimers, etc.,polyimine(s), such as PEI: poly(ethyleneimine), poly(propyleneimine),etc., polyallylamine, sugar backbone based polymers, such ascyclodextrin based polymers, dextran based polymers, chitosan, etc.,silan backbone based polymers, such as PMOXA-PDMS copolymers, etc.,blockpolymers consisting of a combination of one or more cationic blocks(e.g. selected from a cationic polymer as mentioned above) and of one ormore hydrophilic or hydrophobic blocks (e.g. polyethyleneglycole), etc.

In other embodiments the RNA (e.g., mRNA) vaccine is not associated witha cationic or polycationic compounds.

In some embodiments, a nanoparticle comprises compounds of Formula (I):

or a salt or isomer thereof, wherein:

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR,

—CHQR, —CQ(R)₂, and unsubstituted C₁₋₆ alkyl, where Q is selected from acarbocycle, heterocycle, —OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃,—CX₂H, —CXH₂, —CN, —N(R)₂, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R,—N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂, —N(R)R₈, —O(CH₂)_(n)OR,—N(R)C(═NR₉)N(R)₂, —N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR,—N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR, —N(OR)C(O)N(R)₂,—N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂, —N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)N(R)₂,—C(═NR₉)R, —C(O)N(R)O R, and —C(R)N(R)₂C(O)OR, and each n isindependently selected from 1, 2, 3, 4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,

—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;

R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

In some embodiments, a subset of compounds of Formula (I) includes thosein which when R₄ is —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, or —CQ(R)₂, then(i) Q is not —N(R)₂ when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or7-membered heterocycloalkyl when n is 1 or 2.

In some embodiments, another subset of compounds of Formula (I) includesthose in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR,

—CHQR, —CQ(R)₂, and unsubstituted C₁₋₆ alkyl, where Q is selected from aC₃₋₆ carbocycle, a 5- to 14-membered heteroaryl having one or moreheteroatoms selected from N, O, and S, —OR,—O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —C(O)N(R)₂,—N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂, —CRN(R)₂C(O)OR,—N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂, —N(R)C(═CHR₉)N(R)₂,—OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR,—N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)O R, and a 5- to14-membered heterocycloalkyl having one or more heteroatoms selectedfrom N, O, and S which is substituted with one or more substituentsselected from oxo (═O), OH, amino, mono- or di-alkylamino, and C₁₋₃alkyl, and each n is independently selected from 1, 2, 3, 4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;

R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts orisomers thereof.

In some embodiments, another subset of compounds of Formula (I) includesthose in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to 14-memberedheterocycle having one or more heteroatoms selected from N, O, and S,—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—CRN(R)₂C(O)OR, —N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂,—N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R,—N(OR)C(O)OR, —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and —C(═NR₉)N(R)₂, and eachn is independently selected from 1, 2, 3, 4, and 5; and when Q is a 5-to 14-membered heterocycle and (i) R₄ is —(CH₂)_(n)Q in which n is 1 or2, or (ii) R₄ is —(CH₂)_(n)CHQR in which n is 1, or (iii) R₄ is —CHQR,and —CQ(R)₂, then Q is either a 5- to 14-membered heteroaryl or 8- to14-membered heterocycloalkyl;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;

R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or isomers thereof.

In some embodiments, another subset of compounds of Formula (I) includesthose in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to 14-memberedheteroaryl having one or more heteroatoms selected from N, O, and S,—OR,

—O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —C(O)N(R)₂,—N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂, —CRN(R)₂C(O)OR,—N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂, —N(R)C(═CHR₉)N(R)₂,—OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR,—N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and —C(═NR₉)N(R)₂, and eachn is independently selected from 1, 2, 3, 4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;

R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts orisomers thereof.

In some embodiments, another subset of compounds of Formula (I) includesthose in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₂₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is —(CH₂)_(n)Q or —(CH₂)_(n)CHQR, where Q is —N(R)₂, and n isselected from 3, 4, and 5;

each R5 is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₁₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or isomers thereof.

In some embodiments, another subset of compounds of Formula (I) includesthose in which

R1 is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of C₁₋₁₄alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃, togetherwith the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of —(CH₂)_(n)Q, —(CH₂)_(n)CHQR,—CHQR, and —CQ(R)₂, where Q is —N(R)₂, and n is selected from 1, 2, 3,4, and 5;

each R5 is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

-   -   each R′ is independently selected from the group consisting of        C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₁₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or isomers thereof.

In some embodiments, a subset of compounds of Formula (I) includes thoseof Formula (IA):

or a salt or isomer thereof, wherein 1 is selected from 1, 2, 3, 4, and5; m is selected from 5, 6, 7, 8, and 9; M₁ is a bond or M′; R₄ isunsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q, in which Q is OH,—NHC(S)N(R)₂, —NHC(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)R₈,—NHC(═NR₉)N(R)₂, —NHC(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, heteroarylor heterocycloalkyl; M and M′ are independently selected

from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —S—S—, an aryl group,and a heteroaryl group; and R₂ and R₃ are independently selected fromthe group consisting of H, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.

In some embodiments, a subset of compounds of Formula (I) includes thoseof Formula (II):

or a salt or isomer thereof, wherein 1 is selected from 1, 2, 3, 4, and5; M₁ is a bond or M′; R₄ is unsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q,in which n is 2, 3, or 4, and Q isOH, —NHC(S)N(R)₂, —NHC(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)R₈,—NHC(═NR₉)N(R)₂, —NHC(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, heteroarylor heterocycloalkyl; M and M′ are independently selectedfrom —C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —S—S—, an aryl group,and a heteroaryl group; and R₂ and R₃ are independently selected fromthe group consisting of H, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.

In some embodiments, a subset of compounds of Formula (I) includes thoseof Formula (IIa), (IIb), (IIc), or (IIe):

or a salt or isomer thereof, wherein R₄ is as described herein.

In some embodiments, a subset of compounds of Formula (I) includes thoseof Formula (IId):

or a salt or isomer thereof, wherein n is 2, 3, or 4; and m, R′, R″, andR₂ through R₆ are as described herein. For example, each of R₂ and R₃may be independently selected from the group consisting of C₅₋₁₄ alkyland C₅₋₁₄ alkenyl.

In some embodiments, the compound of Formula (I) is selected from thegroup consisting of:

In further embodiments, the compound of Formula (I) is selected from thegroup consisting of:

In some embodiments, the compound of Formula (I) is selected from thegroup consisting of:

and salts and isomers thereof.

In some embodiments, a nanoparticle comprises the following compound:

or salts and isomers thereof.

In some embodiments, the disclosure features a nanoparticle compositionincluding a lipid component comprising a compound as described herein(e.g., a compound according to Formula (I), (IA), (II), (IIa), (IIb),(IIc), (IId) or (IIe)).

In some embodiments, the disclosure features a pharmaceuticalcomposition comprising a nanoparticle composition according to thepreceding embodiments and a pharmaceutically acceptable carrier. Forexample, the pharmaceutical composition is refrigerated or frozen forstorage and/or shipment (e.g., being stored at a temperature of 4° C. orlower, such as a temperature between about −150° C. and about 0° C. orbetween about −80° C. and about −20° C. (e.g., about −5° C., −10° C.,−15° C., −20° C., −25° C., −30° C., −40° C., −50° C., −60° C., −70° C.,−80° C., −90° C., −130° C. or −150° C.). For example, the pharmaceuticalcomposition is a solution that is refrigerated for storage and/orshipment at, for example, about −20° C., −30° C., −40° C., −50° C., −60°C., −70° C., or −80° C.

In some embodiments, the disclosure provides a method of delivering atherapeutic and/or prophylactic (e.g., RNA, such as mRNA) to a cell(e.g., a mammalian cell). This method includes the step of administeringto a subject (e.g., a mammal, such as a human) a nanoparticlecomposition including (i) a lipid component including a phospholipid(such as a polyunsaturated lipid), a PEG lipid, a structural lipid, anda compound of Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or(IIe) and (ii) a therapeutic and/or prophylactic, in which administeringinvolves contacting the cell with the nanoparticle composition, wherebythe therapeutic and/or prophylactic is delivered to the cell.

In some embodiments, the disclosure provides a method of producing apolypeptide of interest in a cell (e.g., a mammalian cell). The methodincludes the step of contacting the cell with a nanoparticle compositionincluding (i) a lipid component including a phospholipid (such as apolyunsaturated lipid), a PEG lipid, a structural lipid, and a compoundof Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe) and (ii)an mRNA encoding the polypeptide of interest, whereby the mRNA iscapable of being translated in the cell to produce the polypeptide.

In some embodiments, the disclosure provides a method of treating adisease or disorder in a mammal (e.g., a human) in need thereof. Themethod includes the step of administering to the mammal atherapeutically effective amount of a nanoparticle composition including(i) a lipid component including a phospholipid (such as apolyunsaturated lipid), a PEG lipid, a structural lipid, and a compoundof Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe) and (ii)a therapeutic and/or prophylactic (e.g., an mRNA). In some embodiments,the disease or disorder is characterized by dysfunctional or aberrantprotein or polypeptide activity. For example, the disease or disorder isselected from the group consisting of rare diseases, infectiousdiseases, cancer and proliferative diseases, genetic diseases (e.g.,cystic fibrosis), autoimmune diseases, diabetes, neurodegenerativediseases, cardio- and reno-vascular diseases, and metabolic diseases.

In some embodiments, the disclosure provides a method of delivering(e.g., specifically delivering) a therapeutic and/or prophylactic to amammalian organ (e.g., a liver, spleen, lung, or femur). This methodincludes the step of administering to a subject (e.g., a mammal) ananoparticle composition including (i) a lipid component including aphospholipid, a PEG lipid, a structural lipid, and a compound of Formula(I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe) and (ii) atherapeutic and/or prophylactic (e.g., an mRNA), in which administeringinvolves contacting the cell with the nanoparticle composition, wherebythe therapeutic and/or prophylactic is delivered to the target organ(e.g., a liver, spleen, lung, or femur).

In some embodiments, the disclosure features a method for the enhanceddelivery of a therapeutic and/or prophylactic (e.g., an mRNA) to atarget tissue (e.g., a liver, spleen, lung, or femur). This methodincludes administering to a subject (e.g., a mammal) a nanoparticlecomposition, the composition including (i) a lipid component including acompound of Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or(IIe), a phospholipid, a structural lipid, and a PEG lipid; and (ii) atherapeutic and/or prophylactic, the administering including contactingthe target tissue with the nanoparticle composition, whereby thetherapeutic and/or prophylactic is delivered to the target tissue.

In some embodiments, the disclosure features a method of loweringimmunogenicity comprising introducing the nanoparticle composition ofthe disclosure into cells, wherein the nanoparticle composition reducesthe induction of the cellular immune response of the cells to thenanoparticle composition, as compared to the induction of the cellularimmune response in cells induced by a reference composition whichcomprises a reference lipid instead of a compound of Formula (I), (IA),(II), (IIa), (IIb), (IIc), (IId) or (IIe). For example, the cellularimmune response is an innate immune response, an adaptive immuneresponse, or both.

The disclosure also includes methods of synthesizing a compound ofFormula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe) and methodsof making a nanoparticle composition including a lipid componentcomprising the compound of Formula (I), (IA), (II), (IIa), (IIb), (IIc),(IId) or (IIe).

Modes of Vaccine Administration

Tropical disease RNA (e.g. mRNA) vaccines may be administered by anyroute which results in a therapeutically effective outcome. Theseinclude, but are not limited, to intradermal, intramuscular, intranasaland/or subcutaneous administration. The present disclosure providesmethods comprising administering RNA (e.g., mRNA) vaccines to a subjectin need thereof. The exact amount required will vary from subject tosubject, depending on the species, age, and general condition of thesubject, the severity of the disease, the particular composition, itsmode of administration, its mode of activity, and the like. Tropicaldisease RNA (e.g., mRNA) vaccines compositions are typically formulatedin dosage unit form for ease of administration and uniformity of dosage.It will be understood, however, that the total daily usage of RNA (e.g.,mRNA) vaccine compositions may be decided by the attending physicianwithin the scope of sound medical judgment. The specific therapeuticallyeffective, prophylactically effective, or appropriate imaging dose levelfor any particular patient will depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;the activity of the specific compound employed; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration, route of administration, and rateof excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coincidental with the specificcompound employed; and like factors well known in the medical arts.

In some embodiments, tropical disease RNA (e.g. mRNA) vaccinescompositions may be administered at dosage levels sufficient to deliver0.0001 mg/kg to 100 mg/kg, 0.001 mg/kg to 0.05 mg/kg, 0.005 mg/kg to0.05 mg/kg, 0.001 mg/kg to 0.005 mg/kg, 0.05 mg/kg to 0.5 mg/kg, 0.01mg/kg to 50 mg/kg, 0.1 mg/kg to 40 mg/kg, 0.5 mg/kg to 30 mg/kg, 0.01mg/kg to 10 mg/kg, 0.1 mg/kg to 10 mg/kg, or 1 mg/kg to 25 mg/kg, ofsubject body weight per day, one or more times a day, per week, permonth, etc. to obtain the desired therapeutic, diagnostic, prophylactic,or imaging effect (see, e.g., the range of unit doses described inInternational Publication No WO2013078199, the contents of which areherein incorporated by reference in their entirety). The desired dosagemay be delivered three times a day, two times a day, once a day, everyother day, every third day, every week, every two weeks, every threeweeks, every four weeks, every 2 months, every three months, every 6months, etc. In some embodiments, the desired dosage may be deliveredusing multiple administrations (e.g., two, three, four, five, six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or moreadministrations). When multiple administrations are employed, splitdosing regimens such as those described herein may be used. In exemplaryembodiments, tropical disease RNA (e.g., mRNA) vaccines compositions maybe administered at dosage levels sufficient to deliver 0.0005 mg/kg to0.01 mg/kg, e.g., about 0.0005 mg/kg to about 0.0075 mg/kg, e.g., about0.0005 mg/kg, about 0.001 mg/kg, about 0.002 mg/kg, about 0.003 mg/kg,about 0.004 mg/kg or about 0.005 mg/kg.

In some embodiments, tropical disease RNA (e.g., mRNA) vaccinecompositions may be administered once or twice (or more) at dosagelevels sufficient to deliver 0.025 mg/kg to 0.250 mg/kg, 0.025 mg/kg to0.500 mg/kg, 0.025 mg/kg to 0.750 mg/kg, or 0.025 mg/kg to 1.0 mg/kg.

In some embodiments, tropical disease RNA (e.g., mRNA) vaccinecompositions may be administered twice (e.g., Day 0 and Day 7, Day 0 andDay 14, Day 0 and Day 21, Day 0 and Day 28, Day 0 and Day 60, Day 0 andDay 90, Day 0 and Day 120, Day 0 and Day 150, Day 0 and Day 180, Day 0and 3 months later, Day 0 and 6 months later, Day 0 and 9 months later,Day 0 and 12 months later, Day 0 and 18 months later, Day 0 and 2 yearslater, Day 0 and 5 years later, or Day 0 and 10 years later) at a totaldose of or at dosage levels sufficient to deliver a total dose of 0.0100mg, 0.025 mg, 0.050 mg, 0.075 mg, 0.100 mg, 0.125 mg, 0.150 mg, 0.175mg, 0.200 mg, 0.225 mg, 0.250 mg, 0.275 mg, 0.300 mg, 0.325 mg, 0.350mg, 0.375 mg, 0.400 mg, 0.425 mg, 0.450 mg, 0.475 mg, 0.500 mg, 0.525mg, 0.550 mg, 0.575 mg, 0.600 mg, 0.625 mg, 0.650 mg, 0.675 mg, 0.700mg, 0.725 mg, 0.750 mg, 0.775 mg, 0.800 mg, 0.825 mg, 0.850 mg, 0.875mg, 0.900 mg, 0.925 mg, 0.950 mg, 0.975 mg, or 1.0 mg. Higher and lowerdosages and frequency of administration are encompassed by the presentdisclosure. For example, a tropical disease RNA (e.g., mRNA) vaccinecomposition may be administered three or four times.

In some embodiments, tropical disease RNA (e.g., mRNA) vaccinecompositions may be administered twice (e.g., Day 0 and Day 7, Day 0 andDay 14, Day 0 and Day 21, Day 0 and Day 28, Day 0 and Day 60, Day 0 andDay 90, Day 0 and Day 120, Day 0 and Day 150, Day 0 and Day 180, Day 0and 3 months later, Day 0 and 6 months later, Day 0 and 9 months later,Day 0 and 12 months later, Day 0 and 18 months later, Day 0 and 2 yearslater, Day 0 and 5 years later, or Day 0 and 10 years later) at a totaldose of or at dosage levels sufficient to deliver a total dose of 0.010mg, 0.025 mg, 0.100 mg or 0.400 mg.

In some embodiments, the tropical disease RNA (e.g., mRNA) vaccine foruse in a method of vaccinating a subject is administered to the subjectas a single dosage of between 10 μg/kg and 400 μg/kg of the nucleic acidvaccine (in an effective amount to vaccinate the subject). In someembodiments the RNA (e.g., mRNA) vaccine for use in a method ofvaccinating a subject is administered to the subject as a single dosageof between 10 μg and 400 μg of the nucleic acid vaccine (in an effectiveamount to vaccinate the subject). In some embodiments, a tropicaldisease RNA (e.g., mRNA) vaccine for use in a method of vaccinating asubject is administered to the subject as a single dosage of 25-1000 μg(e.g., a single dosage of mRNA encoding Malaria (e.g., P. falciparum, P.vivax, P. malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV,DENV, ZIKV and/or YFV antigen). In some embodiments, a tropical diseaseRNA (e.g., mRNA) vaccine is administered to the subject as a singledosage of 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 950 or 1000 μg. For example, a tropicaldisease RNA (e.g., mRNA) vaccine may be administered to a subject as asingle dose of 25-100, 25-500, 50-100, 50-500, 50-1000, 100-500,100-1000, 250-500, 250-1000, or 500-1000 μg. In some embodiments, atropical disease RNA (e.g., mRNA) vaccine for use in a method ofvaccinating a subject is administered to the subject as two dosages, thecombination of which equals 25-1000 μg of the tropical disease RNA(e.g., mRNA) vaccine.

A tropical disease RNA (e.g. mRNA) vaccine pharmaceutical compositiondescribed herein can be formulated into a dosage form described herein,such as an intranasal, intratracheal, or injectable (e.g., intravenous,intraocular, intravitreal, intramuscular, intradermal, intracardiac,intraperitoneal, intranasal and subcutaneous).

Tropical Disease RNA (e.g., mRNA) Vaccine Formulations and Methods ofUse

Some aspects of the present disclosure provide formulations of thetropical disease RNA (e.g., mRNA) vaccine, wherein the RNA (e.g., mRNA)vaccine is formulated in an effective amount to produce an antigenspecific immune response in a subject (e.g., production of antibodiesspecific to an Malaria (e.g., P. falciparum, P. vivax, P. malariaeand/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/orYFV antigenic polypeptide). An “effective amount” is a dose of an RNA(e.g., mRNA) vaccine effective to produce an antigen-specific immuneresponse. Also provided herein are methods of inducing anantigen-specific immune response in a subject.

In some embodiments, the antigen-specific immune response ischaracterized by measuring an anti-Malaria (e.g., P. falciparum, P.vivax, P. malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV,DENV, ZIKV and/or YFV antigenic polypeptide antibody titer produced in asubject administered a tropical disease RNA (e.g., mRNA) vaccine asprovided herein. An antibody titer is a measurement of the amount ofantibodies within a subject, for example, antibodies that are specificto a particular antigen (e.g., an Malaria (e.g., P. falciparum, P.vivax, P. malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV,DENV, ZIKV and/or YFV antigenic polypeptide) or epitope of an antigen.Antibody titer is typically expressed as the inverse of the greatestdilution that provides a positive result. Enzyme-linked immunosorbentassay (ELISA) is a common assay for determining antibody titers, forexample.

In some embodiments, an antibody titer is used to assess whether asubject has had an infection or to determine whether immunizations arerequired. In some embodiments, an antibody titer is used to determinethe strength of an autoimmune response, to determine whether a boosterimmunization is needed, to determine whether a previous vaccine waseffective, and to identify any recent or prior infections. In accordancewith the present disclosure, an antibody titer may be used to determinethe strength of an immune response induced in a subject by the tropicaldisease RNA (e.g., mRNA) vaccine.

In some embodiments, an anti-antigenic polypeptide (e.g., ananti-Malaria (e.g., P. falciparum, P. vivax, P. malariae and/or P.ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFVantigenic polypeptide) antibody titer produced in a subject is increasedby at least 1 log relative to a control. For example, anti-antigenicpolypeptide antibody titer produced in a subject may be increased by atleast 1.5, at least 2, at least 2.5, or at least 3 log relative to acontrol. In some embodiments, the anti-antigenic polypeptide antibodytiter produced in the subject is increased by 1, 1.5, 2, 2.5 or 3 logrelative to a control. In some embodiments, the anti-antigenicpolypeptide antibody titer produced in the subject is increased by 1-3log relative to a control. For example, the anti-antigenic polypeptideantibody titer produced in a subject may be increased by 1-1.5, 1-2,1-2.5, 1-3, 1.5-2, 1.5-2.5, 1.5-3, 2-2.5, 2-3, or 2.5-3 log relative toa control.

In some embodiments, the anti-antigenic polypeptide (e.g., ananti-Malaria (e.g., P. falciparum, P. vivax, P. malariae and/or P.ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFVantigenic polypeptide) antibody titer produced in a subject is increasedat least 2 times relative to a control. For example, the anti-antigenicpolypeptide antibody titer produced in a subject may be increased atleast 3 times, at least 4 times, at least 5 times, at least 6 times, atleast 7 times, at least 8 times, at least 9 times, or at least 10 timesrelative to a control. In some embodiments, the anti-antigenicpolypeptide antibody titer produced in the subject is increased 2, 3, 4,5,6, 7, 8, 9, or 10 times relative to a control. In some embodiments,the anti-antigenic polypeptide antibody titer produced in a subject isincreased 2-10 times relative to a control. For example, theanti-antigenic polypeptide antibody titer produced in a subject may beincreased 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7,3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6,6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 times relativeto a control.

A control, in some embodiments, is the anti-antigenic polypeptide (e.g.,an anti-Malaria (e.g., P. falciparum, P. vivax, P. malariae and/or P.ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFVantigenic polypeptide) antibody titer produced in a subject who has notbeen administered a tropical disease RNA (e.g., mRNA) vaccine of thepresent disclosure. In some embodiments, a control is an anti-antigenicpolypeptide (e.g., an anti-Malaria (e.g., P. falciparum, P. vivax, P.malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKVand/or YFV antigenic polypeptide) antibody titer produced in a subjectwho has been administered a live attenuated Malaria (e.g., P.falciparum, P. vivax, P. malariae and/or P. ovale), JEV, WNV, EEEV,VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV vaccine. An attenuated vaccineis a vaccine produced by reducing the virulence of a viable (live)virus. An attenuated virus is altered in a manner that renders itharmless or less virulent relative to a live, unmodified virus. In someembodiments, a control is an anti-antigenic polypeptide (e.g., ananti-Malaria (e.g., P. falciparum, P. vivax, P. malariae and/or P.ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFVantigenic polypeptide) antibody titer produced in a subject administeredinactivated Malaria (e.g., P. falciparum, P. vivax, P. malariae and/orP. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFVvaccine. In some embodiments, a control is an anti-antigenic polypeptide(e.g., an anti-Malaria (e.g., P. falciparum, P. vivax, P. malariaeand/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/orYFV antigenic polypeptide) antibody titer produced in a subjectadministered a recombinant or purified Malaria (e.g., P. falciparum, P.vivax, P. malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV,DENV, ZIKV and/or YFV protein vaccine. Recombinant protein vaccinestypically include protein antigens that either have been produced in aheterologous expression system (e.g., bacteria or yeast) or purifiedfrom large amounts of the pathogenic organism. In some embodiments, acontrol is an anti-antigenic polypeptide (e.g., an anti-Malaria (e.g.,P. falciparum, P. vivax, P. malariae and/or P. ovale), JEV, WNV, EEEV,VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV antigenic polypeptide) antibodytiter produced in a subject who has been administered an Malaria (e.g.,P. falciparum, P. vivax, P. malariae and/or P. ovale), JEV, WNV, EEEV,VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV virus-like particle (VLP)vaccine.

In some embodiments, an effective amount of a tropical disease RNA(e.g., mRNA) vaccine is a dose that is reduced compared to the standardof care dose of a recombinant Malaria (e.g., P. falciparum, P. vivax, P.malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKVand/or YFV protein vaccine. A “standard of care,” as provided herein,refers to a medical or psychological treatment guideline and can begeneral or specific. “Standard of care” specifies appropriate treatmentbased on scientific evidence and collaboration between medicalprofessionals involved in the treatment of a given condition. It is thediagnostic and treatment process that a physician/clinician shouldfollow for a certain type of patient, illness or clinical circumstance.A “standard of care dose,” as provided herein, refers to the dose of arecombinant or purified Malaria (e.g., P. falciparum, P. vivax, P.malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKVand/or YFV protein vaccine, or a live attenuated or inactivated Malaria(e.g., P. falciparum, P. vivax, P. malariae and/or P. ovale), JEV, WNV,EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV vaccine, that aphysician/clinician or other medical professional would administer to asubject to treat or prevent Malaria (e.g., P. falciparum, P. vivax, P.malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKVand/or YFV, or a related condition, while following the standard of careguideline for treating or preventing Malaria (e.g., P. falciparum, P.vivax, P. malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV,DENV, ZIKV and/or YFV, or a related condition.

In some embodiments, the anti-antigenic polypeptide (e.g., ananti-Malaria (e.g., P. falciparum, P. vivax, P. malariae and/or P.ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFVantigenic polypeptide) antibody titer produced in a subject administeredan effective amount of a tropical disease RNA (e.g., mRNA) vaccine isequivalent to an anti-antigenic polypeptide (e.g., an anti-Malaria(e.g., P. falciparum, P. vivax, P. malariae and/or P. ovale), JEV, WNV,EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV antigenic polypeptide)antibody titer produced in a control subject administered a standard ofcare dose of a recombinant or purified Malaria (e.g., P. falciparum, P.vivax, P. malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV,DENV, ZIKV and/or YFV protein vaccine or a live attenuated orinactivated Malaria (e.g., P. falciparum, P. vivax, P. malariae and/orP. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFVvaccine.

In some embodiments, an effective amount of a tropical disease RNA(e.g., mRNA) vaccine is a dose equivalent to an at least 2-foldreduction in a standard of care dose of a recombinant or purifiedMalaria (e.g., P. falciparum, P. vivax, P. malariae and/or P. ovale),JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV proteinvaccine. For example, an effective amount of a tropical disease RNA(e.g., mRNA) vaccine may be a dose equivalent to an at least 3-fold, atleast 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, atleast 8-fold, at least 9-fold, or at least 10-fold reduction in astandard of care dose of a recombinant or purified Malaria (e.g., P.falciparum, P. vivax, P. malariae and/or P. ovale), JEV, WNV, EEEV,VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV protein vaccine. In someembodiments, an effective amount of a tropical disease RNA (e.g., mRNA)vaccine is a dose equivalent to an at least at least 100-fold, at least500-fold, or at least 1000-fold reduction in a standard of care dose ofa recombinant or purified Malaria (e.g., P. falciparum, P. vivax, P.malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKVand/or YFV protein vaccine. In some embodiments, an effective amount ofa tropical disease RNA (e.g., mRNA) vaccine is a dose equivalent to a2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 20-, 50-, 100-, 250-, 500-, or1000-fold reduction in a standard of care dose of a recombinant orpurified Malaria (e.g., P. falciparum, P. vivax, P. malariae and/or P.ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV proteinvaccine. In some embodiments, the anti-antigenic polypeptide antibodytiter produced in a subject administered an effective amount of atropical disease RNA (e.g., mRNA) vaccine is equivalent to ananti-antigenic polypeptide antibody titer produced in a control subjectadministered the standard of care dose of a recombinant or proteinMalaria (e.g., P. falciparum, P. vivax, P. malariae and/or P. ovale),JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV protein vaccineor a live attenuated or inactivated Malaria (e.g., P. falciparum, P.vivax, P. malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV,DENV, ZIKV and/or YFV vaccine. In some embodiments, an effective amountof a tropical disease RNA (e.g., mRNA) vaccine is a dose equivalent to a2-fold to 1000-fold (e.g., 2-fold to 100-fold, 10-fold to 1000-fold)reduction in the standard of care dose of a recombinant or purifiedMalaria (e.g., P. falciparum, P. vivax, P. malariae and/or P. ovale),JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV proteinvaccine, wherein the anti-antigenic polypeptide antibody titer producedin the subject is equivalent to an anti-antigenic polypeptide antibodytiter produced in a control subject administered the standard of caredose of a recombinant or purified Malaria (e.g., P. falciparum, P.vivax, P. malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV,DENV, ZIKV and/or YFV protein vaccine or a live attenuated orinactivated Malaria (e.g., P. falciparum, P. vivax, P. malariae and/orP. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFVvaccine.

In some embodiments, the effective amount of a tropical disease RNA(e.g., mRNA) vaccine is a dose equivalent to a 2 to 1000-, 2 to 900-, 2to 800-, 2 to 700-, 2 to 600-, 2 to 500-, 2 to 400-, 2 to 300-, 2 to200-, 2 to 100-, 2 to 90-, 2 to 80-, 2 to 70-, 2 to 60-, 2 to 50-, 2 to40-, 2 to 30-, 2 to 20-, 2 to 10-, 2 to 9-, 2 to 8-, 2 to 7-, 2 to 6-, 2to 5-, 2 to 4-, 2 to 3-, 3 to 1000-, 3 to 900-, 3 to 800-, 3 to 700-, 3to 600-, 3 to 500-, 3 to 400-, 3 to 3 to 00-, 3 to 200-, 3 to 100-, 3 to90-, 3 to 80-, 3 to 70-, 3 to 60-, 3 to 50-, 3 to 40-, 3 to 30-, 3 to20-, 3 to 10-, 3 to 9-, 3 to 8-, 3 to 7-, 3 to 6-, 3 to 5-, 3 to 4-, 4to 1000-, 4 to 900-, 4 to 800-, 4 to 700-, 4 to 600-, 4 to 500-, 4 to400-, 4 to 4 to 00-, 4 to 200-, 4 to 100-, 4 to 90-, 4 to 80-, 4 to 70-,4 to 60-, 4 to 50-, 4 to 40-, 4 to 30-, 4 to 20-, 4 to 10-, 4 to 9-, 4to 8-, 4 to 7-, 4 to 6-, 4 to 5-, 4 to 4-, 5 to 1000-, 5 to 900-, 5 to800-, 5 to 700-, 5 to 600-, 5 to 500-, 5 to 400-, 5 to 300-, 5 to 200-,5 to 100-, 5 to 90-, 5 to 80-, 5 to 70-, 5 to 60-, 5 to 50-, 5 to 40-, 5to 30-, 5 to 20-, 5 to 10-, 5 to 9-, 5 to 8-, 5 to 7-, 5 to 6-, 6 to1000-, 6 to 900-, 6 to 800-, 6 to 700-, 6 to 600-, 6 to 500-, 6 to 400-,6 to 300-, 6 to 200-, 6 to 100-, 6 to 90-, 6 to 80-, 6 to 70-, 6 to 60-,6 to 50-, 6 to 40-, 6 to 30-, 6 to 20-, 6 to 10-, 6 to 9-, 6 to 8-, 6 to7-, 7 to 1000-, 7 to 900-, 7 to 800-, 7 to 700-, 7 to 600-, 7 to 500-, 7to 400-, 7 to 300-, 7 to 200-, 7 to 100-, 7 to 90-, 7 to 80-, 7 to 70-,7 to 60-, 7 to 50-, 7 to 40-, 7 to 30-, 7 to 20-, 7 to 10-, 7 to 9-, 7to 8-, 8 to 1000-, 8 to 900-, 8 to 800-, 8 to 700-, 8 to 600-, 8 to500-, 8 to 400-, 8 to 300-, 8 to 200-, 8 to 100-, 8 to 90-, 8 to 80-, 8to 70-, 8 to 60-, 8 to 50-, 8 to 40-, 8 to 30-, 8 to 20-, 8 to 10-, 8 to9-, 9 to 1000-, 9 to 900-, 9 to 800-, 9 to 700-, 9 to 600-, 9 to 500-, 9to 400-, 9 to 300-, 9 to 200-, 9 to 100-, 9 to 90-, 9 to 80-, 9 to 70-,9 to 60-, 9 to 50-, 9 to 40-, 9 to 30-, 9 to 20-, 9 to 10-, 10 to 1000-,10 to 900-, 10 to 800-, 10 to 700-, 10 to 600-, 10 to 500-, 10 to 400-,10 to 300-, 10 to 200-, 10 to 100-, 10 to 90-, 10 to 80-, 10 to 70-, 10to 60-, 10 to 50-, 10 to 40-, 10 to 30-, 10 to 20-, 20 to 1000-, 20 to900-, 20 to 800-, 20 to 700-, 20 to 600-, 20 to 500-, 20 to 400-, 20 to300-, 20 to 200-, 20 to 100-, 20 to 90-, 20 to 80-, 20 to 70-, 20 to60-, 20 to 50-, 20 to 40-, 20 to 30-, 30 to 1000-, 30 to 900-, 30 to800-, 30 to 700-, 30 to 600-, 30 to 500-, 30 to 400-, 30 to 300-, 30 to200-, 30 to 100-, 30 to 90-, 30 to 80-, 30 to 70-, 30 to 60-, 30 to 50-,30 to 40-, 40 to 1000-, 40 to 900-, 40 to 800-, 40 to 700-, 40 to 600-,40 to 500-, 40 to 400-, 40 to 300-, 40 to 200-, 40 to 100-, 40 to 90-,40 to 80-, 40 to 70-, 40 to 60-, 40 to 50-, 50 to 1000-, 50 to 900-, 50to 800-, 50 to 700-, 50 to 600-, 50 to 500-, 50 to 400-, 50 to 300-, 50to 200-, 50 to 100-, 50 to 90-, 50 to 80-, 50 to 70-, 50 to 60-, 60 to1000-, 60 to 900-, 60 to 800-, 60 to 700-, 60 to 600-, 60 to 500-, 60 to400-, 60 to 300-, 60 to 200-, 60 to 100-, 60 to 90-, 60 to 80-, 60 to70-, 70 to 1000-, 70 to 900-, 70 to 800-, 70 to 700-, 70 to 600-, 70 to500-, 70 to 400-, 70 to 300-, 70 to 200-, 70 to 100-, 70 to 90-, 70 to80-, 80 to 1000-, 80 to 900-, 80 to 800-, 80 to 700-, 80 to 600-, 80 to500-, 80 to 400-, 80 to 300-, 80 to 200-, 80 to 100-, 80 to 90-, 90 to1000-, 90 to 900-, 90 to 800-, 90 to 700-, 90 to 600-, 90 to 500-, 90 to400-, 90 to 300-, 90 to 200-, 90 to 100-, 100 to 1000-, 100 to 900-, 100to 800-, 100 to 700-, 100 to 600-, 100 to 500-, 100 to 400-, 100 to300-, 100 to 200-, 200 to 1000-, 200 to 900-, 200 to 800-, 200 to 700-,200 to 600-, 200 to 500-, 200 to 400-, 200 to 300-, 300 to 1000-, 300 to900-, 300 to 800-, 300 to 700-, 300 to 600-, 300 to 500-, 300 to 400-,400 to 1000-, 400 to 900-, 400 to 800-, 400 to 700-, 400 to 600-, 400 to500-, 500 to 1000-, 500 to 900-, 500 to 800-, 500 to 700-, 500 to 600-,600 to 1000-, 600 to 900-, 600 to 800-, 600 to 700-, 700 to 1000-, 700to 900-, 700 to 800-, 800 to 1000-, 800 to 900-, or 900 to 1000-foldreduction in the standard of care dose of a recombinant Malaria (e.g.,P. falciparum, P. vivax, P. malariae and/or P. ovale), JEV, WNV, EEEV,VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV protein vaccine. In someembodiments, the anti-antigenic polypeptide antibody titer produced inthe subject is equivalent to an anti-antigenic polypeptide antibodytiter produced in a control subject administered the standard of caredose of a recombinant or purified Malaria (e.g., P. falciparum, P.vivax, P. malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV,DENV, ZIKV and/or YFV protein vaccine or a live attenuated orinactivated Malaria (e.g., P. falciparum, P. vivax, P. malariae and/orP. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFVvaccine. In some embodiments, the effective amount is a dose equivalentto (or equivalent to an at least) 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-,20-, 30-, 40-, 50-, 60-, 70-, 80-, 90-, 100-, 110-, 120-, 130-, 140-,150-, 160-, 170-, 1280-, 190-, 200-, 210-, 220-, 230-, 240-, 250-, 260-,270-, 280-, 290-, 300-, 310-, 320-, 330-, 340-, 350-, 360-, 370-, 380-,390-, 400-, 410-, 420-, 430-, 440-, 450-, 4360-, 470-, 480-, 490-, 500-,510-, 520-, 530-, 540-, 550-, 560-, 5760-, 580-, 590-, 600-, 610-, 620-,630-, 640-, 650-, 660-, 670-, 680-, 690-, 700-, 710-, 720-, 730-, 740-,750-, 760-, 770-, 780-, 790-, 800-, 810-, 820-, 830-, 840-, 850-, 860-,870-, 880-, 890-, 900-, 910-, 920-, 930-, 940-, 950-, 960-, 970-, 980-,990-, or 1000-fold reduction in the standard of care dose of arecombinant Malaria (e.g., P. falciparum, P. vivax, P. malariae and/orP. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFVprotein vaccine. In some embodiments, an anti-antigenic polypeptideantibody titer produced in the subject is equivalent to ananti-antigenic polypeptide antibody titer produced in a control subjectadministered the standard of care dose of a recombinant or purifiedMalaria (e.g., P. falciparum, P. vivax, P. malariae and/or P. ovale),JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV protein vaccineor a live attenuated or inactivated Malaria (e.g., P. falciparum, P.vivax, P. malariae and/or P. ovale), JEV, WNV, EEEV, VEEV, SINV, CHIKV,DENV, ZIKV and/or YFV vaccine.

In some embodiments, the effective amount of a tropical disease RNA(e.g., mRNA) vaccine is a total dose of 50-1000 μg. In some embodiments,the effective amount of a tropical disease RNA (e.g., mRNA) vaccine is atotal dose of 50-1000, 50-900, 50-800, 50-700, 50-600, 50-500, 50-400,50-300, 50-200, 50-100, 50-90, 50-80, 50-70, 50-60, 60-1000, 60-900,60-800, 60-700, 60-600, 60-500, 60-400, 60-300, 60-200, 60-100, 60-90,60-80, 60-70, 70-1000, 70-900, 70-800, 70-700, 70-600, 70-500, 70-400,70-300, 70-200, 70-100, 70-90, 70-80, 80-1000, 80-900, 80-800, 80-700,80-600, 80-500, 80-400, 80-300, 80-200, 80-100, 80-90, 90-1000, 90-900,90-800, 90-700, 90-600, 90-500, 90-400, 90-300, 90-200, 90-100,100-1000, 100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300,100-200, 200-1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400,200-300, 300-1000, 300-900, 300-800, 300-700, 300-600, 300-500, 300-400,400-1000, 400-900, 400-800, 400-700, 400-600, 400-500, 500-1000,500-900, 500-800, 500-700, 500-600, 600-1000, 600-900, 600-900, 600-700,700-1000, 700-900, 700-800, 800-1000, 800-900, or 900-1000 μg. In someembodiments, the effective amount of a tropical disease RNA (e.g., mRNA)vaccine is a total dose of 50, 100, 150, 200, 250, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 μg. In someembodiments, the effective amount is a dose of 25-500 μg administered tothe subject a total of two times. In some embodiments, the effectiveamount of a tropical disease RNA (e.g., mRNA) vaccine is a dose of25-500, 25-400, 25-300, 25-200, 25-100, 25-50, 50-500, 50-400, 50-300,50-200, 50-100, 100-500, 100-400, 100-300, 100-200, 150-500, 150-400,150-300, 150-200, 200-500, 200-400, 200-300, 250-500, 250-400, 250-300,300-500, 300-400, 350-500, 350-400, 400-500 or 450-500 μg administeredto the subject a total of two times. In some embodiments, the effectiveamount of a tropical disease RNA (e.g., mRNA) vaccine is a total dose of25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 μg administeredto the subject a total of two times.

Examples of Additional Embodiments of the Disclosure

1. A tropical disease vaccine, comprising:

at least one messenger ribonucleic acid (mRNA) polynucleotide having a5′ terminal cap, an open reading frame encoding at least one tropicaldisease antigenic polypeptide, and a 3′ polyA tail.

2. The vaccine of paragraph 1, wherein the at least one tropical diseaseantigenic polypeptide is selected from a Malaria (e.g., P. falciparum,P. vivax, P. malariae and/or P. ovale) antigenic polypeptide, a JEVantigenic polypeptide, a WNV antigenic polypeptide, a EEEV antigenicpolypeptide, a VEEV antigenic polypeptide, a SINV antigenic polypeptide,a CHIKV antigenic polypeptide, a DENV antigenic polypeptide, a ZIKVantigenic polypeptide and a YFV antigenic polypeptide.3. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide is encoded by a sequence identified by any one of SEQ IDNO: 1-6, 18, 19, 30-34, 48, 49, 55, 56, 65-80, 118-136, 223-239 or376-388, or a fragment of a sequence identified by any one of SEQ ID NO:1-6, 18, 19, 30-34, 48, 49, 55, 56, 65-80, 118-136, 223-239 or 376-388.4. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide comprises a sequence identified by any one of SEQ ID NO:7-12, 20-21, 35-39, 50-51, 57-58, 81-96, 137-155, 240-256, or 389-401,or a fragment of a sequence identified by any one of SEQ ID NO: 7-12,20-21, 35-39, 50-51, 57-58, 81-96, 137-155, 240-256, or 389-401.5. The vaccine of paragraph 1, wherein the at least one antigenicpolypeptide comprises a sequence identified by any one of SEQ ID NO:13-17, 22-29, 44-47, 52-54, 59-64, 97-117, 156-222, 469, 259-291 or402-413, or a fragment of a sequence identified by any one of SEQ ID NO:13-17, 22-29, 44-47, 52-54, 59-64, 97-117, 156-222, 469, 259-291 or402-413.6. The vaccine of any one of paragraphs 1-5, wherein the 5′ terminal capis or comprises 7 mG(5′)ppp(5′)NlmpNp.7. The vaccine of any one of paragraphs 1-6, wherein 100% of the uracilin the open reading frame is modified to include N1-methyl pseudouridineat the 5-position of the uracil.8. The vaccine of any one of paragraphs 1-7, wherein the vaccine isformulated in a lipid nanoparticle comprising: DLin-MC3-DMA;cholesterol; 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC); andpolyethylene glycol (PEG)2000-DMG.9. The vaccine of paragraph 8, wherein the lipid nanoparticle furthercomprises trisodium citrate buffer, sucrose and water.

This disclosure is not limited in its application to the details ofconstruction and the arrangement of components set forth in thefollowing description or illustrated in the drawings. The disclosure iscapable of other embodiments and of being practiced or of being carriedout in various ways. Also, the phraseology and terminology used hereinis for the purpose of description and should not be regarded aslimiting. The use of “including,” “comprising,” or “having,”“containing,” “involving,” and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

EXAMPLES Example 1: Manufacture of Polynucleotides

According to the present disclosure, the manufacture of polynucleotidesand/or parts or regions thereof may be accomplished utilizing themethods taught in International Publication WO2014/152027, entitled“Manufacturing Methods for Production of RNA Transcripts,” the contentsof which is incorporated herein by reference in its entirety.

Purification methods may include those taught in InternationalPublication WO2014/152030 and International Publication WO2014/152031,each of which is incorporated herein by reference in its entirety.

Detection and characterization methods of the polynucleotides may beperformed as taught in International Publication WO2014/144039, which isincorporated herein by reference in its entirety.

Characterization of the polynucleotides of the disclosure may beaccomplished using polynucleotide mapping, reverse transcriptasesequencing, charge distribution analysis, detection of RNA impurities,or any combination of two or more of the foregoing. “Characterizing”comprises determining the RNA transcript sequence, determining thepurity of the RNA transcript, or determining the charge heterogeneity ofthe RNA transcript, for example. Such methods are taught in, forexample, International Publication WO2014/144711 and InternationalPublication WO2014/144767, the content of each of which is incorporatedherein by reference in its entirety.

Example 2: Chimeric Polynucleotide Synthesis

According to the present disclosure, two regions or parts of a chimericpolynucleotide may be joined or ligated using triphosphate chemistry. Afirst region or part of 100 nucleotides or less is chemicallysynthesized with a 5′ monophosphate and terminal 3′desOH or blocked OH,for example. If the region is longer than 80 nucleotides, it may besynthesized as two strands for ligation.

If the first region or part is synthesized as a non-positionallymodified region or part using in vitro transcription (IVT), conversionthe 5′monophosphate with subsequent capping of the 3′ terminus mayfollow.

Monophosphate protecting groups may be selected from any of those knownin the art.

The second region or part of the chimeric polynucleotide may besynthesized using either chemical synthesis or IVT methods. IVT methodsmay include an RNA polymerase that can utilize a primer with a modifiedcap. Alternatively, a cap of up to 130 nucleotides may be chemicallysynthesized and coupled to the IVT region or part.

For ligation methods, ligation with DNA T4 ligase, followed by treatmentwith DNase should readily avoid concatenation.

The entire chimeric polynucleotide need not be manufactured with aphosphate-sugar backbone. If one of the regions or parts encodes apolypeptide, then such region or part may comprise a phosphate-sugarbackbone.

Ligation is then performed using any known click chemistry, orthoclickchemistry, solulink, or other bioconjugate chemistries known to those inthe art.

Synthetic Route

The chimeric polynucleotide may be made using a series of startingsegments. Such segments include:

(a) a capped and protected 5′ segment comprising a normal 3′OH (SEG. 1)

(b) a 5′ triphosphate segment, which may include the coding region of apolypeptide and a normal 3′OH (SEG. 2)

(c) a 5′ monophosphate segment for the 3′ end of the chimericpolynucleotide (e.g., the tail) comprising cordycepin or no 3′OH (SEG.3)

After synthesis (chemical or IVT), segment 3 (SEG. 3) may be treatedwith cordycepin and then with pyrophosphatase to create the 5′monophosphate.

Segment 2 (SEG. 2) may then be ligated to SEG. 3 using RNA ligase. Theligated polynucleotide is then purified and treated with pyrophosphataseto cleave the diphosphate. The treated SEG. 2-SEG. 3 construct may thenbe purified and SEG. 1 is ligated to the 5′ terminus. A furtherpurification step of the chimeric polynucleotide may be performed.

Where the chimeric polynucleotide encodes a polypeptide, the ligated orjoined segments may be represented as: 5′UTR (SEG. 1), open readingframe or ORF (SEG. 2) and 3′UTR+PolyA (SEG. 3).

The yields of each step may be as much as 90-95%.

Example 3: PCR for cDNA Production

PCR procedures for the preparation of cDNA may be performed using 2×KAPAHIFI™ HotStart ReadyMix by Kapa Biosystems (Woburn, Mass.). This systemincludes 2×KAPA ReadyMix 12.5 μl; Forward Primer (10 μM) 0.75 μl;Reverse Primer (10 μM) 0.75 μl; Template cDNA 100 ng; and dH₂0 dilutedto 25.0 μl. The reaction conditions may be at 95° C. for 5 min. Thereaction may be performed for 25 cycles of 98° C. for 20 sec, then 58°C. for 15 sec, then 72° C. for 45 sec, then 72° C. for 5 min, then 4° C.to termination.

The reaction may be cleaned up using Invitrogen's PURELINK™ PCR MicroKit (Carlsbad, Calif.) per manufacturer's instructions (up to 5 μg).Larger reactions may require a cleanup using a product with a largercapacity. Following the cleanup, the cDNA may be quantified using theNANODROP™ and analyzed by agarose gel electrophoresis to confirm thatthe cDNA is the expected size. The cDNA may then be submitted forsequencing analysis before proceeding to the in vitro transcriptionreaction.

Example 4: In Vitro Transcription (IVT)

The in vitro transcription reaction generates RNA polynucleotides. Suchpolynucleotides may comprise a region or part of the polynucleotides ofthe disclosure, including chemically modified RNA (e.g., mRNA)polynucleotides. The chemically modified RNA polynucleotides can beuniformly modified polynucleotides. The in vitro transcription reactionutilizes a custom mix of nucleotide triphosphates (NTPs). The NTPs maycomprise chemically modified NTPs, or a mix of natural and chemicallymodified NTPs, or natural NTPs.

A typical in vitro transcription reaction includes the following:

1) Template cDNA 1.0 μg 2) 10x transcription buffer 2.0 μl (400 mMTris-HCl pH 8.0, 190 mM MgCl₂, 50 mM DTT, 10 mM Spermidine) 3) CustomNTPs (25 mM each) 0.2 μl 4) RNase Inhibitor 20 U 5) T7 RNA polymerase3000 U 6) dH₂0 up to 20.0 μl. and 7) Incubation at 37° C. for 3 hr-5hrs.

The crude IVT mix may be stored at 4° C. overnight for cleanup the nextday. 1 U of RNase-free DNase may then be used to digest the originaltemplate. After 15 minutes of incubation at 37° C., the mRNA may bepurified using Ambion's MEGACLEAR™ Kit (Austin, Tex.) following themanufacturer's instructions. This kit can purify up to 500 μg of RNA.Following the cleanup, the RNA polynucleotide may be quantified usingthe NANODROP™ and analyzed by agarose gel electrophoresis to confirm theRNA polynucleotide is the proper size and that no degradation of the RNAhas occurred.

Example 5: Enzymatic Capping

Capping of a RNA polynucleotide is performed as follows where themixture includes: IVT RNA 60 μg-180 μg and dH₂0 up to 72 μl. The mixtureis incubated at 65° C. for 5 minutes to denature RNA, and then istransferred immediately to ice.

The protocol then involves the mixing of 10× Capping Buffer (0.5 MTris-HCl (pH 8.0), 60 mM KCl, 12.5 mM MgCl₂) (10.0 μl); 20 mM GTP (5.0μl); 20 mM S-Adenosyl Methionine (2.5 μl); RNase Inhibitor (100 U);2′-O-Methyltransferase (400U); Vaccinia capping enzyme (Guanylyltransferase) (40 U); dH₂0 (Up to 28 μl); and incubation at 37° C. for 30minutes for 60 μg RNA or up to 2 hours for 180 μg of RNA.

The RNA polynucleotide may then be purified using Ambion's MEGACLEAR™Kit (Austin, Tex.) following the manufacturer's instructions. Followingthe cleanup, the RNA may be quantified using the NANODROP™(ThermoFisher, Waltham, Mass.) and analyzed by agarose gelelectrophoresis to confirm the RNA polynucleotide is the proper size andthat no degradation of the RNA has occurred. The RNA polynucleotideproduct may also be sequenced by running a reverse-transcription-PCR togenerate the cDNA for sequencing.

Example 6: PolyA Tailing Reaction

Without a poly-T in the cDNA, a poly-A tailing reaction must beperformed before cleaning the final product. This is done by mixingcapped IVT RNA (100 μl); RNase Inhibitor (20 U); 10× Tailing Buffer (0.5M Tris-HCl (pH 8.0), 2.5 M NaCl, 100 mM MgCl₂) (12.0 μl); 20 mM ATP (6.0μl); Poly-A Polymerase (20 U); dH₂0 up to 123.5 μl and incubation at 37°C. for 30 min. If the poly-A tail is already in the transcript, then thetailing reaction may be skipped and proceed directly to cleanup withAmbion's MEGACLEAR™ kit (Austin, Tex.) (up to 500 μg). Poly-A Polymerasemay be a recombinant enzyme expressed in yeast.

It should be understood that the processivity or integrity of the polyAtailing reaction may not always result in an exact size polyA tail.Hence, polyA tails of approximately between 40-200 nucleotides, e.g.,about 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 150-165, 155, 156,157, 158, 159, 160, 161, 162, 163, 164 or 165 are within the scope ofthe present disclosure.

Example 7: Natural 5′ Caps and 5′ Cap Analogues

5′-capping of polynucleotides may be completed concomitantly during thein vitro-transcription reaction using the following chemical RNA capanalogs to generate the 5′-guanosine cap structure according tomanufacturer protocols: 3′-O-Me-m7G(5′)ppp(5′) G [the ARCA cap];G(5′)ppp(5′)A; G(5′)ppp(5′)G; m7G(5′)ppp(5′)A; m7G(5′)ppp(5′)G (NewEngland BioLabs, Ipswich, Mass.). 5′-capping of modified RNA may becompleted post-transcriptionally using a Vaccinia Virus Capping Enzymeto generate the “Cap 0” structure: m7G(5′)ppp(5′)G (New England BioLabs,Ipswich, Mass.). Cap 1 structure may be generated using both VacciniaVirus Capping Enzyme and a 2′-O methyl-transferase to generate:m7G(5′)ppp(5′)G-2′-O-methyl. Cap 2 structure may be generated from theCap 1 structure followed by the 2′-O-methylation of the5′-antepenultimate nucleotide using a 2′-O methyl-transferase. Cap 3structure may be generated from the Cap 2 structure followed by the2′-O-methylation of the 5′-preantepenultimate nucleotide using a 2′-Omethyl-transferase. Enzymes are preferably derived from a recombinantsource.

When transfected into mammalian cells, the modified mRNAs have astability of between 12-18 hours or more than 18 hours, e.g., 24, 36,48, 60, 72 or greater than 72 hours.

Example 8: Capping Assays Protein Expression Assay

Polynucleotides (e.g., mRNA) encoding a polypeptide, containing any ofthe caps taught herein, can be transfected into cells at equalconcentrations. The amount of protein secreted into the culture mediumcan be assayed by ELISA at 6, 12, 24 and/or 36 hours post-transfection.Synthetic polynucleotides that secrete higher levels of protein into themedium correspond to a synthetic polynucleotide with a highertranslationally-competent cap structure.

Purity Analysis Synthesis

RNA (e.g., mRNA) polynucleotides encoding a polypeptide, containing anyof the caps taught herein can be compared for purity using denaturingAgarose-Urea gel electrophoresis or HPLC analysis. RNA polynucleotideswith a single, consolidated band by electrophoresis correspond to thehigher purity product compared to polynucleotides with multiple bands orstreaking bands. Chemically modified RNA polynucleotides with a singleHPLC peak also correspond to a higher purity product. The cappingreaction with a higher efficiency provides a more pure polynucleotidepopulation.

Cytokine Analysis

RNA (e.g., mRNA) polynucleotides encoding a polypeptide, containing anyof the caps taught herein can be transfected into cells at multipleconcentrations. The amount of pro-inflammatory cytokines, such asTNF-alpha and IFN-beta, secreted into the culture medium can be assayedby ELISA at 6, 12, 24 and/or 36 hours post-transfection. RNApolynucleotides resulting in the secretion of higher levels ofpro-inflammatory cytokines into the medium correspond to apolynucleotides containing an immune-activating cap structure.

Capping Reaction Efficiency

RNA (e.g., mRNA) polynucleotides encoding a polypeptide, containing anyof the caps taught herein can be analyzed for capping reactionefficiency by LC-MS after nuclease treatment. Nuclease treatment ofcapped polynucleotides yield a mixture of free nucleotides and thecapped 5′-5-triphosphate cap structure detectable by LC-MS. The amountof capped product on the LC-MS spectra can be expressed as a percent oftotal polynucleotide from the reaction and correspond to cappingreaction efficiency. The cap structure with a higher capping reactionefficiency has a higher amount of capped product by LC-MS.

Example 9: Agarose Gel Electrophoresis of Modified RNA or RT PCRProducts

Individual RNA polynucleotides (200-400 ng in a 20 μl volume) or reversetranscribed PCR products (200-400 ng) may be loaded into a well on anon-denaturing 1.2% Agarose E-Gel (Invitrogen, Carlsbad, Calif.) and runfor 12-15 minutes, according to the manufacturer protocol.

Example 10: NANODROP™ Modified RNA Quantification and UV Spectral Data

Chemically modified RNA polynucleotides in TE buffer (1 μl) are used forNANODROP™ UV absorbance readings to quantitate the yield of eachpolynucleotide from an chemical synthesis or in vitro transcriptionreaction.

Example 11: Formulation of Modified mRNA Using Lipidoids

RNA (e.g., mRNA) polynucleotides may be formulated for in vitroexperiments by mixing the polynucleotides with the lipidoid at a setratio prior to addition to cells. In vivo formulation may require theaddition of extra ingredients to facilitate circulation throughout thebody. To test the ability of these lipidoids to form particles suitablefor in vivo work, a standard formulation process used for siRNA-lipidoidformulations may be used as a starting point. After formation of theparticle, polynucleotide is added and allowed to integrate with thecomplex. The encapsulation efficiency is determined using a standard dyeexclusion assays.

Example 12: Immunogenicity Study

The instant study is designed to test the immunogenicity in mice ofcandidate Malaria vaccines comprising a mRNA polynucleotide encoding CSprotein, LSA1, MSP1, AMA1, TRAP or a combination thereof obtained fromPlasmodium.

Mice are immunized intramuscularly (IM), or intradermally (ID) with mRNAencoding CS protein, LSA1, MSP1, TRAP and AMA1. Up to threeimmunizations are given at 3-week intervals (i.e., at weeks 0,3, and 6),and sera are collected after each immunization until weeks 33-51. Serumantibody titers against CS protein, LSA1, MSP1 and AMA1 are determinedby ELISA. Responses against Plasmodium sporozoites, asexual blood-stageparasites, and gametocytes were determined by using an indirectimmunofluorescence assay (IFA). T cell responses were analyzed byElispot using splenocytes from immunized mice and stimulated withpeptide pools from the relevant antigens.

Example 13: Plasmodium Non-Human Primate Challenge

The instant study is designed to test the efficacy in simians ofcandidate Malaria vaccines against a lethal challenge using a Malariavaccine comprising mRNA encoding CS protein, LSA1, MSP1, AMA1, TRAP or acombination thereof obtained from Plasmodium. Simians are challengedwith a lethal dose of Plasmodium.

Simians are immunized intramuscularly (IM) or intradermally (ID) at week0, week 3 and week 6 with candidate Malaria vaccines.

Serum antibody titers against CS protein, LSA1, MSP1 and AMA1 aredetermined by ELISA. Responses against Plasmodium sporozoites, asexualblood-stage parasites, and gametocytes were determined by using anindirect immunofluorescence assay (IFA). T cell responses were analyzedby Elispot using PBMCs from immunized primates and stimulated withpeptide pools from the relevant antigens.

In experiments where a lipid nanoparticle (LNP) formulation is used, theformulation may include a cationic lipid, non-cationic lipid, PEG lipidand structural lipid in the ratios 50:10:1.5:38.5. The cationic lipidmay be DLin-KC2-DMA (50 mol %), the non-cationic lipid may be DSPC (10mol %), the PEG lipid may be PEG-DOMG (1.5 mol %) and the structurallipid may be cholesterol (38.5 mol %), for example.

Example 14: Plasmodium Human Challenge

The instant study is designed to test the efficacy in human subjects ofcandidate Malaria vaccines against an attenuated challenge (ControlledHuman Malaria Infection (CHMI) Study) using a Malaria vaccine comprisingmRNA encoding CS protein, LSA1, MSP1, AMA1, TRAP or a combinationthereof obtained from Plasmodium. Subjects are challenged with anattenuated (non-lethal) dose of Plasmodium.

Subjects are immunized intramuscularly (IM) or intradermally (ID) atweek 0 and week 3 with candidate Malaria vaccines. Serum is tested formicroneutralization (see Example 16). The subjects are then challengedwith an attenuated dose of Plasmodium on week 7 via IV, IM or ID.Endpoint is day 13 post infection. Body temperature and weight areassessed and recorded daily.

In experiments where a lipid nanoparticle (LNP) formulation is used, theformulation may include a cationic lipid, non-cationic lipid, PEG lipidand structural lipid in the ratios 50:10:1.5:38.5. The cationic lipidmay be DLin-KC2-DMA (50 mol %), the non-cationic lipid may be DSPC (10mol %), the PEG lipid may be PEG-DOMG (1.5 mol %) and the structurallipid may be cholesterol (38.5 mol %), for example.

Example 15: Microneutralization Assay

Nine serial 2-fold dilutions (1:50-1:12,800) of simian or human serumare made in 50 μl virus growth medium (VGM) with trypsin in 96 wellmicrotiter plates. Fifty microliters of Plasmodium are added to theserum dilutions and allowed to incubate for 60 minutes at roomtemperature (RT). Positive control wells of Plasmodium without sera andnegative control wells without Plasmodium or sera are included intriplicate on each plate. While the serum-Plasmodium mixtures incubate,a single cell suspension of cells is prepared by trypsinizing (Gibco0.5% bovine pancrease trypsin in EDTA) a confluent monolayer, andsuspended cells are transferred to a 50 ml centrifuge tube, topped withsterile PBS and gently mixed. The cells are then pelleted at 200 g for 5minutes, supernatant aspirated and cells resuspended in PBS. Thisprocedure is repeated once and the cells are resuspended at aconcentration of 3×10⁵/ml in VGM with porcine trypsin. Then, 100 μl ofcells are added to the serum-virus mixtures and the plates incubated at35° C. in CO₂ for 5 days. The plates are fixed with 80% acetone inphosphate buffered saline (PBS) for 15 minutes at RT, air dried and thenblocked for 30 minutes containing PBS with 0.5% gelatin and 2% FCS. Anantibody to CS protein, LSA1, MSP1, AMA1 or TRAP is diluted in PBS with0.5% gelatin/2% FCS/0.5% Tween 20 and incubated at RT for 2 hours. Wellsare washed and horse radish peroxidase conjugated goat anti-mouse IgGadded, followed by another 2 hour incubation. After washing,O-phenylenediamine dihydrochloride is added and the neutralization titeris defined as the titer of serum that reduced color development by 50%compared to the positive control wells.

Example 16: JEV Immunogenicity Study

This study was designed to test the immunogenicity of JEV prME mRNAvaccines in Balb/c mice. Mice were 6-8 weeks old.

Mice were immunized intramuscularly at three different doses (10 μg, 2μg and 0.5 m). All mice were given two doses of the vaccine, one at day0 and another at day 28. Serum was collected at days 0 and 56, and aplaque reduction neutralization test was used to quantify neutralizingantibody titer. The concentration of serum to reduce the number ofplaques in the assay by 50%, compared to the serum free virus, denotedas PRNT50 was used as a measure of neutralizing antibodies and level ofprotection against virus.

Results of this study is shown in FIG. 1. A PRNT50 titer of greater than1:10 is considered protective. JEV mRNA vaccine at 10 μg doses resultsin a very high titer, indicative of a high potency vaccine.

Example 17: Immunogenicity Cross-Neutralization Study

The instant study is designed to test the immunogenicity andcross-neutralization in mice of candidate combination vaccinescomprising a mRNA polynucleotide encoding antigenic polypeptides (e.g.,envelope proteins) obtained from Plasmodium, JEV, WNV, EEEV, VEEV, SINV,CHIKV, DENV, ZIKV and/or YFV.

Mice are immunized intravenously (IV), intramuscularly (IM), orintradermally (ID) with candidate combination vaccines. A total of fourimmunizations are given at 3-week intervals (at weeks 0, 3, 6, and 9),and sera are collected after each immunization until weeks 33-51. Serumantibody titers against envelope proteins are determined by ELISA. Seracollected from each mouse during weeks 10-16 are pooled, and total IgGsare purified by using ammonium sulfate (Sigma) precipitation followed byDEAE (Pierce) batch purification. Following dialysis against PBS, thepurified antibodies are used for immunoelectron microscopy,antibody-affinity testing, and an in vitro protection assay.

Example 18: Immunogenicity Studies for Combination RNA Vaccine

BALB/C mice are immunized with mRNA encoded Plasmodium, JEV, WNV, EEEV,VEEV, SINV, CHIKV, DENV, ZIKV and/or YFV antigenic polypeptides, forexample, as shown in Table 7 below and according to the followingdosing/bleeding schedule: prime dose on day 0, boost dose on day 28,bleeding on days 0, 28, 42 and 56.

The mice are administered a combination vaccine, combining two or moreof the Plasmodium, JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV, ZIKV and/orYFV antigenic polypeptides, such that all possible combinations aretested. Animals are challenged at day 56 with a second dose of any oneof the antigenic polypeptides included in the original dose.

An efficacy study using mRNA encoded West Nile prMEs and JapaneseEncephalitis prMEs antigens is also performed according to the followingschedule:

Non-human primates are also immunized with mRNA encoded antigen usingsimilar schedules shown in Tables 7 and 8. The animals are tested forimmunogenicity to Plasmodium, JEV, WNV, EEEV, VEEV, SINV, CHIKV, DENV,ZIKV and/or YFV and combinations thereof.

Example 19: YFV Immunogenicity Studies

The instant study is designed to test the immunogenicity in Balb/c miceof candidate yellow fever virus (YFV) vaccines comprising a mRNApolynucleotide encoding YFV prME. Four groups of Balb/c mice (n=5) areimmunized intramuscularly (IM) with 10 μg (n=2) or 2 μg (n=2) of thecandidate vaccine. One group of mice is administered PBS intramuscularlyas a control. All mice are administered an initial dose of vaccine(Groups 1-4) or PBS (Group 5) on Day 0, and then the mice in Groups 1and 3 are administered a boost dose on Day 21, while the mice in Group 5are administered PBS on Day 21. All mice are bled on Day 41. SeeTable 1. Anti-Yellow fever neutralization IgG titer is determined on Day−1, Day 28 and Day 41.

Example 20: YFV Rodent Challenge

The instant study is designed to test the efficacy in AG129 mice ofcandidate yellow fever virus (YFV) vaccines against a lethal challengeusing a YFV vaccine comprising mRNA encoding YFV prME. Four groups ofAG129 mice (n=8) are immunized intramuscularly (IM) with 10 μg (n=2) or2 μg (n=2) of the candidate vaccine. One group of mice is administeredPBS intramuscularly as a control. All mice are administered an initialdose of vaccine (Groups 1-4) or PBS (Group 5) on Day 0, and then themice in Groups 1 and 3 are administered a boost dose on Day 21, whilethe mice in Group 5 are administered PBS on Day 21. All mice arechallenged with a lethal dose of YFV in Day 42. All mice are thenmonitored for survival and weight loss. Anti-Yellow fever neutralizationIgG titer is determined on Day −1, Day 28 and Day 41, and viral load isdetermined 5 days post challenge.

Example 21: Expression of ZIKV prME Protein in Mammalian Cells UsingZIKV mRNA Vaccine Construct

The Zika virus (ZIKV) prME mRNA vaccine construct were tested inmammalian cells (239T cells) for the expression of ZIKV prME protein.293T cells were plated in 24-well plates and were transfected with 2 μgof ZIKV prME mRNA using a Lipofectamine transfection reagent. The cellswere incubated for the expression of the ZIKV prME proteins before theywere lysed in an immunoprecipitation buffer containing proteaseinhibitor cocktails. Reducing agent was not added to the lysis buffer toensure that the cellular proteins were in a non-reduced state. Celllysates were centrifuged at 8,000×g for 20 mins to collect lysed cellprecipitate. The cell precipitates were then stained with anti ZIKVhuman serum and goat anti-human Alexa Fluor 647. Fluorescence wasdetected as an indication of prME expression.

The expression of ZIKV prME protein was also detected byfluorescence-activated cell sorting (FACS) using a flow cytometer. 293Fcells (2×10⁶ cells/ml, 30 ml) were transfected with 120 μg PEI, 1 ml of150 mM NaCl, and 60 μg prME mRNA. Transfected cells were incubated for48 hours at 37° C. in a shaker at 130 rpm and under 5% CO₂. The cellswere then washed with PBS buffer containing 2% FBS and fixed in afixation buffer (PBS buffer containing formalin) for 20 minutes at roomtemperature. The fixed cells were permeabilized in a permeabilizationbuffer (PBS+1% Triton X100+1 μl of Golgi plug/ml of cells). Thepermeabilized cells were then stained with anti-ZIKV human serum (1:20dilution) and goat anti-human Alexa Fluor 647 secondary antibody, beforethey were sorted on a flow cytometer. As shown in FIG. 2, FIG. 3A andFIG. 3B, cells transfected with prME mRNA and stained with the anti-ZIKAhuman serum shifted to higher fluorescent intensity, indicating thatprME expressed from the ZIKV mRNA vaccine constructs in the transfectedcells.

Example 22: Expression, Purification and Characterization of ZIKV VLPs

Zika virus (ZIKV) virus-like particles (VLPs) were made in HeLa cellsand in HEK293T cells and purified via PEG precipitation orultracentrifugation, respectively. Cells were cultured in culture media.Prior to transfection, cells were passaged twice in virus growth mediaplus 10% fetal bovine serum (FBS) to media adaptation.

Cells were seeded the day before transfection into T-175 flask. 100 μgof prME-encoding mRNA was transfected using 100 μg pf lipofectamine asper manufacturer's protocol. 6 hours post transfection, monolayers werewashed twice with 1×PBS and 20 mL of virus growth media was added.Supernatant was collected 24-48 hours post transfection bycentrifugation at 2000×g for 10 mins and 0.22 μm filtration.

For VLP purification via PEG precipitation, VLP's were concentratedusing Biovision PEG precipitation kit as per manufacturer's protocol. Inbrief, supernatant with VLP's was mixed with PEG8000 and incubated at 4°C. for 16 hours. After incubation, mixture was centrifuged at 3000×g for30 mins. Pellet containing concentrated VLP's was collected andsuspended into PBS. VLP's were further buffer exchanged into PBS (1:500)using amicon ultra 100 MWCO filter. Purified samples were negativestained to show the presence of assembled VLP particles.

Expression of prME from the vaccine mRNA constructs was demonstrated toresult in the production of virus like particles (VLPs) that areexpected to present to the immune system as identical to Zika virusparticles. Negative stain electron micrographs of supernatants from HeLacells transfected with mRNA encoding Zika prME showed that thevirus-like particles (VLPs), purified by PEG precipitation, have highlyuniform size (˜35-40 nm) and morphology. The bumpy appearance of the VLPsurface appears to reflect mostly immature morphology due to expressionfrom HeLa cells, which have very low expression of furin, a hostprotease that is required for maturation the viral envelope. Uponmaturation, these VLPs will have an exterior structure essentiallyidentical to wild type viral particles, thus eliciting a broad immuneresponse to future Zika virus exposure.

For VLP purification via ultracentrifugation, 293T cells weretransfected with Zika prME mRNA as described herein. Supernatant wascollected 24 hours after changing the media as described herein (30hours post transfection). VLPs were concentrated using Biovision PEGvirus precipitation kit into 500 μL volume. VLPs were further purifiedusing a 10-50% sucrose gradient. Sample layer was seen between 20-30%sucrose layers and collected. VLPs were buffered exchanged into PBS by1:1000 dilution using a 100 MWCO amicon ultra filter. VLPs wereconcentrated after PEG precipitation, and ultracentrifuge-purified VLPswere analyzed for purity on a reducing SDS-PAGE gel (FIG. 4).

Example 23: ZIKV mRNA Vaccine Immunogenicity Studies

The instant study was designed to test the immunogenicity in Balb/c miceof candidate ZIKV vaccines comprising a mRNA polynucleotide encodingZIKV prME. Four groups of Balb/c mice (n=5) were immunizedintramuscularly (IM) with 10 μg (n=2) or 2 μg (n=2) of the candidatevaccine. One group of mice was administered PBS intramuscularly as acontrol. All mice were administered an initial dose of vaccine (Groups1-4) or PBS (Group 5) on Day 0, and then the mice in Groups 1 and 3 wereadministered a boost dose on Day 21, while the mice in Group 5 wereadministered PBS on Day 21. All mice were bled on Day 41. See Table 29.Anti-Zika neutralization IgG titer was determined on Day −1, Day 28 andDay 41 (FIG. 5).

Day 42 neutralizing titers reached EC50s of 427 for 2 μg and 690 for 10μg. The control serum in this experiment was from naturally infectedimmunocompromised mice (Ifnar1−/−, derived from B/6 lineage) in whichhigh viral loads would be achieved.

Example 24: ZIKV Rodent Challenge

The instant study was designed to test the efficacy in AG129 mice ofcandidate ZIKV vaccines against a lethal challenge using a ZIKV vaccinecomprising mRNA encoding ZIKV prME. Four groups of AG129 mice (n=8) wereimmunized intramuscularly (IM) with 10 μg (n=2) or 2 μg (n=2) of thecandidate vaccine. One group of mice was administered PBSintramuscularly as a control. All mice were administered an initial doseof vaccine (Groups 1-4) or PBS (Group 5) on Day 0, and then the mice inGroups 1 and 3 were administered a boost dose on Day 21, while the micein Group 5 were administered PBS on Day 21. All mice were challengedwith a lethal dose of ZIKV in Day 42. All mice were then monitored forsurvival and weight loss. Anti-Zika neutralization IgG titer wasdetermined on Day −1, Day 28 and Day 41, and viral load was determined 5days post challenge. The 10 μg dose provided 100% protection, even witha single dose, and the 2 μg dose provided 60% protection with a singledose and 90% protection with prime-boost doses (see FIGS. 7A and 7B).

In experiments where a lipid nanoparticle (LNP) formulation is used, theformulation may include a cationic lipid, non-cationic lipid, PEG lipidand structural lipid in the ratios 50:10:1.5:38.5. The cationic lipidmay be DLin-KC2-DMA or DLin-MC3-DMA (50 mol %), the non-cationic lipidmay be DSPC (10 mol %), the PEG lipid is PEG-DOMG or PEG-DMG (1.5 mol %)and the structural lipid may be cholesterol (38.5 mol %), for example.

Example 25: Exemplary Dengue Sequences

An exemplary Dengue virus (DENY) peptide epitope may include two or moreepitopes. The epitopes can be of the same sequence or different sequenceand can be all T-cell epitopes, all B-cell epitopes or a combination ofboth. Furthermore, various end units for enhancing MHC processing of thepeptides are possible.

The following sequences represent exemplary DENY peptide epitopesidentified using a database screen (the sequences correspond to SEQ IDNO: 357-360):

DenV1 1

80 DenV2 1

80 DenV3 1

80 DenV4 1

80 DenV1 81

160 DenV2 81

160 DenV3 81

158 DenV4 81

160 DenV1 161

240 DenV2 161

240 DenV3 159

238 DenV4 161

240 DenV1 241

320 DenV2 241

320 DenV3 239

318 DenV4 241

320 DenV1 321

400 DenV2 321

394 DenV3 319

392 DenV4 321

400

Nucleic acid and amino acid sequences for each of DENV-1, DENV-2,DENV-3, and DENV-4 are shown in Tables 28 and 29, respectively.

Example 26: Dengue Virus RNA Vaccine Immunogenicity in Mice

This study provides a preliminary analysis of the immunogenicity of anucleic acid mRNA vaccine using a Dengue virus (DENV) serotype 2 antigenin BALB/c mice. The study utilizes 44 groups of 10 BALB/c female (5) andmale (5) mice (440 total, 6-8 weeks of age at study initiation, seeTable 10 for design summary). In this study, construct numbers used arereferenced and found in Table 28.

Mice were vaccinated on weeks 0 and 3 via intramuscular (IM) orintradermal (ID) routes. One group remained unvaccinated and one wasadministered 10⁵ plaque-forming units (PFU) live DENV2, D2Y98P isolatevia intravenous (IV) injection as a positive control. Serum wascollected from each mouse on weeks 1, 3, and 5; bleeds on weeks 1 and 3were in-life samples (tail vein or submandibular bleeds) and week 5 willbe a terminal (intracardiac) bleed. Individual serum samples were storedat −80° C. until analysis by neutralization or microneutralizationassay. Pooled samples from each group at the week 5 time points weretested by Western blot for reactivity with viral lysate.

Signal was detected in groups 5, 15, 39, and 44 (live virus control) bya band that appeared between 50 and 60 kDa in the Western blot data. Thedata suggests that a mRNA vaccine to a single dengue viral antigen canproduce antibody in preliminary studies.

In order to provide a Dengue vaccine having enhanced immunogenicity, RNAvaccines for concatemeric antigens were designed and tested according tothe invention. These vaccines, which have significantly enhancedactivity, in comparison to the single protein antigens described herein,are described below.

Example 27: in Silico Prediction of T Cell Epitopes for RNA VaccineDesign

Several peptide epitopes from Dengue virus were generated and tested forantigenic activity. The peptide epitopes are designed to maximize MHCpresentation. In general the process of MHC class I presentation isquite inefficient, with only 1 peptide of 10,000 degraded moleculesactually being presented. Additionally the priming of CD8 T cell withAPCs having insufficient densities of surface peptide/MHC class Icomplexes results in weak responders exhibiting impaired cytokinesecretion and a decrease memory pool. Thus, the process of designinghighly effective peptide epitopes is important to the immunogenicity ofthe ultimate vaccine.

In silico prediction of desirable peptide epitopes was performed usingImmune Epitope Database. Using this database several immunogenic DengueT cell epitopes showing strong homology across all 4 Dengue serotypeswere predicted. Examples of these epitopes are shown in FIGS. 8A-8C and9A-9C.

Example 28: Prediction of DENV T Cell Epitopes for RNA Vaccine Design

The design of optimized vaccination systems to prevent or treatconditions that have failed to respond to more traditional treatments orearly vaccination strategies relies on the identification of theantigens or epitopes that play a role in these conditions and which theimmune system can effectively target. T cell epitopes (e.g., MHC peptidebinding) for the various alleles shown in Table 32 were determined usingRapid Epitope Discovery System (ProImmune REVEAL & ProVE®—see Tables33-40 for peptides). This system is used to identify those candidateepitopes that actually cause relevant immune responses from the numerousother potential candidates identified using algorithms to predictMHC-peptide binding. The REVEAL binding assay determines the ability ofeach candidate peptide to bind to one or more MHC I class alleles andstabilize the MHC-peptide complex. The assay identifies the most likelyimmunogenic peptides in a protein sequence by comparing the binding tothat of a high affinity T cell epitope and detecting the presence orabsence of the native conformation of the MHC-peptide complex. Theepitope peptides are further tested using the assays described herein toconfirm their immunogenic activity.

Example 29: Activity Testing for Predicted Peptide Epitopes

Exemplary peptide epitopes selected using the methods described abovewere further characterized. These peptide epitopes were confirmed tohave activity using in vitro HLA binding assays (human lymphocytebinding assays). Peptides (9 aa peptides from the dengue antigen) werescreened for their ability to bind to HLA. The analysis of the homology,affinity, frequency and design of these peptides is shown in FIGS. 8A-8Cand 9A-9C.

Example 30: In Vivo Analysis of Mimectopes of Predicted Human EpitopesRNA Vaccines Methods

IFNγ ELISpot. Mouse IFNγ ELISpot assays were performed using IFNγ coatedMillipore IP Opaque plates according to the manufacturer's mouse IFNγELISPOT guidelines. Briefly, the plates were blocked using complete RPMI(R10) and incubated for 30 minutes prior to plating cells. Peptides(284-292, 408-419 or 540-548) were diluted to 5 different concentrationsfor stimulation at 5, -6, -7, -8, or -9 from an original stockconcentration of 10 mM⁽⁻²⁾. Mouse splenocytes (200,000-250,000 cells)were plated in appropriate wells with peptide, PMA+ Ionomycin or R10media alone. Cells were stimulated in a total volume of 125 μL per well.Plates were then incubated at 37° C., 5% CO₂ for 18-24 hrs. Plates weredeveloped following the manufacturer's instructions. Plates were countedand quality controlled using the automated ELISPOT reader CTLImmunoSpot/FluoroSpot.

Intracellular Cytokine Staining (ICS).

Intracellular Cytokine Staining (ICS). For intracellular cytokinestaining, individual splenocytes, were resuspended at a concentration of1.5×10⁶ cells per mL. Peptides (284-292, 408-419 or 540-548) were madeinto 5 dilutions from a stock concentration of 10 mM⁽⁻²⁾. The finalconcentrations of each peptide were −5, −6, −7, −8, or −9 in theirrespective wells. Cells were stimulated in a final volume of 200 μLwithin a 96 well culture plate. After the addition of Golgi plug (0.2 μLper well), cells were incubated at 37° C., 5% CO₂ for 5 hours. Followingstimulation, cells were surface stained, fixed, washed and put at 4° C.overnight. Intracellular staining was performed the following day,resulting in full panel of Live/Dead (Invitrogen), αCD3, αCD4, αCD8,αCD45, αCCR7, αCD44, αCD25, αIL-2, αIFNγ, and αTNFα (BD Biosciences).Cells were acquired in a 96-U bottom plate using BD LSR Fortessa HTS (BDBiosciences).

Results

The exemplary peptide epitopes selected using the methods describedherein were used to produce tests mouse mimectopes of the predictedhuman epitopes. These mimectopes were analyzed for in vivo activityusing restimulation assays during the acute phase of Dengue infection(Day 7). The methods were performed on dengue-infectedIFNαβ/γ-receptor-deficient mice (AG129). Seven days post infectionsplenocytes were harvested and subjected to an ELISPOT assay to quantifysecretion of cytokines by T cells (CD8) as described above. Briefly, theisolated splenocytes were stimulated with the test peptides and testedfor T cell activation. If the peptide is an appropriate antigen, somecells would be present antigen during infection and would be capable ofstimulating T cells. The methods for analyzing the T cell activationwere performed as follows:

-   -   T cells (at a known concentration) were incubated with a        specific antigen in a cell culture well    -   the activated T cells were transferred to ELISPOT plates        (precoated with anti-cytokine antibody)    -   the cells were incubated such that cytokines could be secreted    -   the cells were washed off the plate and enzyme coupled secondary        Ig was added    -   the plates were washed and substrate was added    -   positive spots were scored under microscope.

The data is shown in FIGS. 10 and 11. FIGS. 10 and 11 are graphsdepicting the results of an ELISPOT assay of dengue-specific peptidesmeasuring IFN-γ (spots per million splenocytes).

A schematic of an assay on a BLT Mouse Model (Bone Marrow/Liver/Thymus)is shown in FIG. 12. The results of a histogram analysis of human CD8 Tcells stimulated with peptide epitope is also shown in FIG. 12.

The following two sequences were used as controls:

(SEQ ID NO: 361) (V5)8-cathb:Kozak Start GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST Stop (SEQ ID NO: 362) (V5)8-cathb +MHCi: Kozak Start GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST StopSome results are shown in Table 41.

Example 31: AG129 Mouse Challenge of Mimectopes of Predicted HumanEpitopes from DENV2

A study is performed on AG129 mouse using a cocktail of 2 peptideepitopes. The immunogenicity of the peptide epitopes is determined inAG129 mice against challenge with a lethal dose of mouse-adapted DENV 2strain D2Y98P. AG129 mice, which lack IFN α/β and γ receptor signaling,injected intradermally in the footpad with 10⁴ PFU of DENV do notsurvive past day 5 post-injection. AG129 mice are vaccinated viaintramuscular (IM) injection with either 2 μg or 10 μg of a cocktail of2 peptide epitopes. The vaccines are given to AG129 mice with a primeand a boost (day 0 and day 28). The positive control group is vaccinatedwith heat-inactivated DENV 2. Phosphate-buffered saline (PBS) is used asa negative control. On day 56, mice are challenged with mouse-adaptedDENV 2 and monitored for 10 days for weight loss, morbidity, andmortality. Mice that display severe illness, defined as >30% weightloss, a health score of 6 or above, extreme lethargy, and/or paralysisare euthanized.

Example 32: “Humanized” DENV Peptides Mouse Immunogenicity Study

A study analyzing immunogenicity of the peptide epitopes on humanizedmice is performed. A single-dose cocktail (30 μg) containing 3 differentpeptide epitopes are delivered by IM route of immunization with primeand boost (day 0, day 28). A T cell (ELISPOT and ICS) characterizationmay be performed on Day 7, Day 28, and Day 56.

Example 33: Testing of Non-Human Primate (NHP) Mimectopes of PredictedDENV Human Epitopes

Non-human primate (NHP) mimectopes to the human epitopes may also bedeveloped and tested for activity in NHP assays. The NHP mimectopes aredesigned based on the human antigen sequence. These mimectopes may beanalyzed for in vivo activity in an NHP model using, for instance,restimulation assays. Once the NHPs have been infected, immune cells maybe isolated and tested for sensitivity of activation by the particularmimectopes.

Example 34: Targeting of DENV Concatemeric Constructs Using CytoplasmicDomain of MHC I

MHC-1_V5 concatemer constructs were developed and transfected in HeLacells. Triple immunofluorescence using Mitotracker Red (mitochondria),anti-V5, and anti-MHC-1 antibodies plus DAPI was performed. MHC-1_V5concatemer transfection in HeLa cells shows V5-MHC1 colocalization.MHC-1 V5 concatemer transfection also shows V5 has homogeneouscytoplasmic distribution and preferentially colocalizes with MHC1 andnot with Mitotracker. These data demonstrate that the V5 concatemer withthe cytoplasmic domain from MHC class I co-localizes with MHC class Iexpression, while the V5 concatemer without this sequence is only foundin the cytoplasm following transfection in HeLa cells.

Example 35: In Vivo Analysis of DENV Concatemeric mRNA Epitope Construct

The Dengue concatemers used in this study consist of 8 repeats of thepeptide TALGATEI (SEQ ID NO: 363), a mouse CD8 T cell epitope found inthe DENV2 envelope. The peptide repeats were linked via cathepsin Bcleavage sites and modified with the various sequences as follows:

-   -   (1) TALGATEI (SEQ ID NO: 363) peptide concatemer with no        modification    -   (2) TALGATEI (SEQ ID NO: 363) peptide concatemer with IgKappa        signal peptide    -   (3) TALGATEI (SEQ ID NO: 363) peptide concatemer with PEST        sequence    -   (4) TALGATEI (SEQ ID NO: 363) peptide concatemer with IgKappa        signal peptide and PEST sequence    -   (5) TALGATEI (SEQ ID NO: 363) peptide concatemer with MHC class        I cytoplasmic domain    -   (6) TALGATEI (SEQ ID NO: 363) peptide concatemer with IgKappa        signal peptide and MHC class I cytoplasmic domain    -   (7) Heat-inactivated DENV2 (D2Y98P)    -   (8) No immunization

The immunogenicity of the peptide concatemeric candidate vaccines wasdetermined in AG129 mice against challenge with a lethal dose of DENVstrain D2Y98P. AG129 mice, which lack IFN α/β and γ receptor signaling,injected intradermally in the footpad with 10⁴ PFU of DENV do notsurvive past day 5 post-injection. (In this study, the mice died due toa problem with the heat-attenuation). The tested vaccines includedconstructs (1)-(8) disclosed above. AG129 mice were vaccinated viaintramuscular (IM) injection with either 2 μg or 10 μg of the candidatevaccine. The vaccines were given to AG129 mice as a prime and a boost(second dose provided 28 days after the first dose). The positivecontrol group was vaccinated with heat-inactivated DENV2.Phosphate-buffered saline (PBS) was used as a negative control.

On day 56, mice were challenged with mouse-adapted DENV2 and monitoredfor 10 days for weight loss, morbidity, and mortality. Mice thatdisplayed severe illness, defined as >30% weight loss, a health score of6 or above, extreme lethargy, and/or paralysis were euthanized. Notably,mice “vaccinated” with heat-inactivated DENV (positive control group)became morbid and died (they were not included in the challenge portionof the study).

In addition, individual serum samples were collected prior to challengeon day 54 and PBMCs were isolated and frozen for subsequent testing.

The AG129 mice PBMCs were thawed and stimulated with TALGATEI (SEQ IDNO: 363) peptide for 5 hours in a standard intracellular cytokine assay.For intracellular cytokine staining, PBMCs were thawed and suspended inmedia. The TALGATEI (SEQ ID NO: 363) peptide was administered tostimulate the cells. After the addition of Golgi plug, cells wereincubated at 37° C., 5% CO₂ for 5 hours. Following stimulation, cellswere surface stained, fixed, washed and put at 4° C. overnight.Intracellular staining was performed the following day and assayed viaELISPOT assay to quantify secretion of cytokines by T cells (CD8) asdescribed above to determine T cell activation. If the peptide were anappropriate antigen, some cells would be present antigen duringinfection and would be capable of stimulating T cells. The results areshown in FIGS. 13A and 13B, which demonstrate that each of the peptides(1)-(6) stimulate T cell activation.

Example 36: Surface-Expressed DENV2 prME Antigens

The DENV2 prME polypeptide antigen sequences provided in Tables 28 and29 were tested to confirm that the DENV prME protein antigen istranslated, properly folded and expressed on the surface of cells. Forthe polypeptide sequences, the bolded sequence is Dengue signalsequence, the underlined sequence is DENV2 precursor membrane sequence,and the unmarked sequence is DENV2 envelope sequence. The sequencesencoding the polypeptides are codon-optimized. HeLa cells weretransfected with DNA encoding the prMEs from nine different DENV2isolates. After 24 hours, surface expression of the prME was detectedusing three different antibodies followed by goat-anti-human AF700secondary antibody and subjecting the cells to FACS analyses. Each ofthe three antibodies is broadly neutralizing DENV2 prME antibodies thathave in vivo efficacy against Dengue virus. D88 binds to DIII ofEnvelope protein for all 4 DENV serotypes (US20150225474). 2D22 binds toDIII of Envelope protein for DENV 2 serotype. 5J7 binds to 3 domains ofEnvelope protein for DENV 3 serotype. FIG. 14B shows that the D88 and2D22 antibodies recognize two of the DENV2 prME antigens. These resultsshow that the two DENV2 prME antigens identified as Thailand/01 68/1979and Peru/IQT29 13/1996 are expressed at the cell surface in aconformationally correct form and are excellent vaccine candidates (FIG.14A). FIG. 14B shows a repeat of staining in triplicate and in twodifferent cell lines (HeLa and 293T). These results confirm properconformation of expressed DENV2 prME antigens (in particular, the prMEantigens from Thailand/01 68/1979 and Peru/IQT29 13/1996) and alsoevidence at least non-inferior and even superior DENV2 antigenicity ascompared to Dengvaxia (CYD-TDV), a live attenuated tetravalent chimericvaccine. Antigen expressed from the mRNA encoding DENV 2 prME fromPeru/IQT2913/1996 shows the best binding to 2 different DENV2 antibodiesin 293T cells and in HeLa cells (D88—binds all 4 serotypes 2D22—bindsDENV 2). This construct has a single amino acid difference from the DENV2 Envelope III Domain immunodeterminant region (see bold, underline inSEQ ID NO: 273, DENV 2 prME (Peru/IQT2913/1996) in Table 29).

Example 37: OVA Multitope In Vitro Screening Assay Kinetic Analysis

Antigen surface presentation is an inefficient process in the antigenpresenting cells (APC). Peptides generated from proteasome degradationof the antigens are presented with low efficiency (only 1 peptide of10000 degraded molecules is actually presented). Thus, priming of CD8 Tcells with APCs provides insufficient densities of surface peptide/MHC Icomplexes, resulting in weak responders exhibiting impaired cytokinesecretion and decreased memory pool. To improve DENV mRNA vaccinesencoding concatemeric DENV antigens, an in vitro assay was designed totest the linkers used to connect peptide repeats, the number of peptiderepeats, and sequences known to enhance antigen presentation.

mRNA constructs encoding one or more OVA epitopes were configured withdifferent linker sequences, protease cleavage sites, and antigenpresentation enhancer sequences. Their respective sequences were asshown in Table 43. To perform the assay, 200 ng of each MC3-formulatedmRNA construct was transfected into JAWSII cells in a 24-well plate.Cells were isolated at 6, 24, and 48 hours post transfection and stainedwith fluorescently-labeled Anti-Mouse OVA257-264 (SIINFEKL (SEQ ID NO:364)) peptide bound to H-2Kb. Staining was analyzed on a LSRFortessaflow cytometer. Samples were run in triplicate. The Mean FluorescentIntensity (MFI) for each mRNA construct was measured and shown in FIG.15. Constructs 2, 3, 7, 9, and 10 showed enhanced surface presentationof the OVA epitope, indicating that the configurations of theseconstructs may be used for DENV mRNA vaccine. Construct 5 comprises asingle OVA peptide and a KDEL sequence that is known to prevent thesecretion of a protein. Construct 5 showed little surface antigenpresentation because the secretion of the peptide was inhibited.

Example 38: Antibody Binding to DENV-1, 2, 3, and 4 prME Epitopes

DENV mRNA vaccines encoding concatemeric antigen epitopes were testedfor binding to antibodies known to recognize one or more DENV serotypes.To test antibody binding to the epitopes, 200 ng of DENV mRNA vaccinesencoding different Dengue prME epitopes were transfected into HeLa cellsin 24-well plates using the TransitIT-mRNA Transfection Kit (Mirus Bio).The DENV mRNA vaccine constructs are shown in Table 28. Transfectionswere done in triplicate. After 24 hours, surface expression was detectedusing four different antibodies (10 μg/mL) followed by eithergoat-anti-human or anti-mouse AF700 secondary antibody (1/500). Signalgenerated from antibody binding are shown as Mean Fluorescent Intensity(MFI) (FIG. 16). Antibody D88 is known to recognize all 4 serotypes andbound to all antigen epitopes encoded by the DENV mRNA vaccineconstructs tested. Antibody 2D22 is known to recognize only DENV 2 andpreferentially bound to construct 21, which encodes DENV 2 antigenepitopes. Antibody 2D22 also showed weak binding to epitopes of otherDENV serotypes. Antibody 5J7 is known to recognize only DENV 3 and onlybound to antigen epitopes encoded by constructs 13, 19, and 20, whichencode DENV 3 antigen epitopes. Antibody 1-11 is known to bind stronglyto DENV 1 and 2, to bind weakly to DENV 3 and to bind little DENV 4.Antibody 1-11 bound to DENV 1, 2, and 3, and binding to DENV 3 antigenepitopes was stronger than binding to DENV 1 or 2 (FIG. 16).

Example 39: DENV prME Challenge Study in Cynomolgus (cyno) Monkey Model

Shown in Table 45 is the design of DENV prME challenge study incynomolgus (cyno) money. Indicated DENV mRNA vaccine encoding prMEantigen epitopes, or vaccines thereof, are used to immunize cyno. Thevaccines are formulated in lipid nanoparticles (e.g., MC3 formulation)and administered to the cyno monkeys intramuscularly on day 0, 21, and42. Dosages of the vaccines are 250 μg or 5 μg per immunization. Inexperiments where a combination of different DENV mRNA vaccines areused, 250 μg or 5 μg of each mRNA vaccine is used. FLAG-tagged H10N8 fluvaccine is used as control at a dosage of 250 μg per immunization. Naïvecyno monkeys without immunization are also used as control. Cyno monkeysera are collected on days 20, 41, 62, and 92 post initial immunizationand used for serotype-specific neutralization assays.

Immunized cyno monkeys are challenged on day 63 post initialimmunization with indicated DENV viruses. Cyno monkey sera are collectedon days 62 (pre-challenge), 63-66, 68, 70, 72, 76, and 92 (end of life)to determine serum viral load.

Example 40: Dengue 2 prME Challenge Study in AG129 Mice

The instant study was designed to evaluate the efficacy of four DENVmRNA vaccine constructs (constructs 21-24 in Table 44) in AG129 micechallenge assays. The schedule of the challenge study is shown in FIG.17A. The DENV mRNA vaccines were formulated in lipid nanoparticles(e.g., MC3 formulation) and administered to the AG129 miceintramuscularly on days 0 and 21. Dosages of the vaccines were 2 μg or10 μg per immunization. Heat inactivated D2Y98P strain was used as anegative control to vaccinate the mice. Naïve AG129 mice withoutimmunization were also used as control.

Immunized AG129 mice were challenged on day 42 post initial immunizationwith Dengue D2Y98P virus (s.c., 1e5 PFU per mouse). AG129 mice sera werecollected on days 20 and 41 post initial immunization and used forserotype-specific neutralization assays. Mice immunized with any of thefour DENV mRNA vaccine constructs survived, while the control mice died.These data demonstrate that, after lethal challenge, there was 100%protection provided by each mRNA vaccine construct, regardless of dose.The weights and health of the mice were monitored and the results wereplotted in FIGS. 17C-17D.

Mice sera collected from mice immunized with 2 μg of the DENV mRNAvaccines were able to neutralize several DENV 2 strains and variationsin the neutralization ability between the tested mRNA vaccines andbetween different DENV 2 strains were observed (FIG. 18).

Example 41: DENV prME Challenge Study in AG129 Mouse Model

Shown in Table 46 is the design of a DENV prME challenge study in AG129mice, including the mRNA constructs tested, the vaccination schedule,the dosage, the challenge strains, and the serum collection schedule.

Indicated DENV mRNA vaccines encoding prME antigen epitopes, or vaccinesthereof, were used to immunize AG129 mice. The vaccines were formulatedin lipid nanoparticles (e.g., MC3 formulation) and administered to themice intramuscularly on days 0 and 21. Dosages of the vaccines were 2 μgor 10 μg per immunization. In experiments where a combination ofdifferent DENV mRNA vaccines was used, 2 μg of each mRNA vaccine wasused. Naïve AG129 mice without immunization were used as control. AG129mice sera were collected on days 20 and 41 post initial immunization andused for serotype-specific neutralization assays.

Immunized AG129 mice were challenged on day 42 post initial immunizationwith Dengue D2Y98P virus (s.c., 1e5 PFU per mouse). The weights andhealth of the mice were monitored for 14 days post infection and theresults were plotted in FIGS. 19A-19I.

Example 42: Virus-Like Particles

The antigens produced from the DENV prME mRNA vaccines of the presentdisclosure, when expressed, are able to assemble into virus-likeparticles (VLPs). The instant study was designed to evaluate theimmunogenicity of the VLPs by negative stain electron microscopeimaging. DENV mRNA vaccine constructs 21-24 were expressed and VLPs wereassembled an isolated. The VLPs were visualized under negative stainelectron microscopy. Construct 23 is the vaccine construct used bySanofi in its DENV vaccines. Constructs 21, 22, and 24 produced moreuniform VLPs, suggesting that these VLPs may be more superior in theirimmunogenicity than the VLPs produced from construct 23.

Example 43: Exemplary Nucleic Acids Encoding CHIKV RNA Polynucleotidesfor Use in a RNA Vaccine

Exemplary sequences that can be used to encode CHIKV E1, E2, E1-E2, andC-E3-E2-6K-E1 RNA polynucleotides for use in the CHIKV RNA vaccine aregiven in Table 47.

Example 44: Protocol to Determine Efficacy of mRNA-Encoded ChikungunyaAntigen Candidates Against CHIKV

Chikungunya virus (CHIKV) has a polycistronic genome and differentantigens, based on the Chikungunya structural protein, are possible.There are membrane-bound and secreted forms of E1 and E2, as well as thefull length polyprotein antigen, which retains the protein's nativeconformation. Additionally, the different CHIKV genotypes can also yielddifferent antigens.

The efficacy of CHIKV candidate vaccines in AG129 mice against challengewith a lethal dose of CHIKV strain 181/25 was investigated. A129 mice,which lack IFN α/β receptor signaling, injected intradermally in thefootpad with 10⁴ PFU of CHIKV 181/25 virus have a 100% survival ratepost-injection. In contrast, AG129 mice, which lack IFN α/β and γreceptor signaling, injected intradermally in the footpad with 10⁴ PFUof CHIKV 181/25 virus do not survive past day 5 post-injection. Thetested vaccines included: MC3-LNP formulated mRNA encoded CHIKV-E1,MC3-LNP formulated mRNA encoded CHIKV-E2, and MC3-LNP formulated mRNAencoded CHIKV-E1/E2/E3/C. Fifteen groups of five AG129 mice werevaccinated via intradermal (ID) or intramuscular (IM) injection witheither 2 μg or 10 μg of the candidate vaccine. The vaccines were givento AG129 mice as single or two doses (second dose provided 28 days afterthe first dose). The positive control group was vaccinated viaintranasal instillation (20 μL volume) with heat-inactivated CHIKV.Phosphate-buffered saline (PBS) was used as a negative control.

On day 56, mice were challenged with 1×10⁴ PFU of CHIKV via ID injectionin a 50 μL volume and monitored for 10 days for weight loss, morbidity,and mortality. Mice that displayed severe illness, defined as >30%weight loss, a health score of 6 or above, extreme lethargy, and/orparalysis were euthanized. Notably, mice “vaccinated” withheat-inactivated CHIKV (positive control group) became morbid and wereeuthanized following the second dose of HI-CHIKV (they were not includedin the challenge portion of the study).

In addition, individual samples were tested for reactivity in asemi-quantitative ELISA for mouse IgG against eitherChikungunya-specific E1 (groups 1-4), Chikungunya-specific E2 (groups5-8), or Chikungunya-specific E1 and E2 proteins (groups 9-15).

The health status is scored as indicated in Table 51.

Example 45: Efficacy of Chikungunya E1 Antigen mRNA Vaccine Candidate

AG129 mice (n=5 per group) were vaccinated with 2 μg or 10 μg ofMC-3-LNP formulated mRNA encoding CHIKV E1. The AG129 mice werevaccinated on either Day 0 or Days 0 and 28 via IM or ID delivery. OnDay 56 following final vaccination all mice were challenged with alethal dose of CHIKV. The survival curve, percent weight loss, andhealth status of the mice vaccinated with 2 μg CHIKV E1 mRNA are shownin FIGS. 21A-21C. The survival results are tabulated in Table 52. Thesurvival curve, percent weight loss, and health status of the micevaccinated with 10 μg CHIKV E1 mRNA are shown in FIGS. 24A-24C. Thesurvival results are tabulated in Table 53.

As shown in Table 52, the 2 μg dose of CHIKV E1 mRNA vaccine gave noprotection post-CHIKV infection challenge when administered via IM or IDwith either a single dose or two doses. Likewise, the single dose of 10μg CHIKV E1 vaccine provided little to no protection when administeredvia IM or ID. However, as indicated in Table 53, the 10 μg dose of CHIKVE1 mRNA vaccine provided 60% protection post-CHIKV challenge whenadministered via IM using two doses and provided 80% protectionpost-CHIKV challenge when administered via ID using two doses.

In all experiments, the negative control mice had a ˜0% survival rate,as did the positive control mice (heat-inactivated CHIKV), which diedbefore CHIKV challenge. Some mice died during the vaccination period.

Example 46: Efficacy of Chikungunya E2 Antigen mRNA Vaccine Candidate

AG129 mice (n=5 per group) were vaccinated with 2 μg or 10 μg ofMC-3-LNP formulated mRNA encoding CHIKV E2. The mice were vaccinated oneither Day 0 or Days 0 and 28 via IM or ID delivery. On Day 56 followingfinal vaccination all mice were challenged with a lethal dose of CHIKV.The survival curve, percent weight loss, and health status of the micevaccinated with 2 μg CHIKV E2 mRNA are shown in FIGS. 22A-22C. Thesurvival results are tabulated in Table 54 below. The survival curve,percent weight loss, and health status of the mice vaccinated with 10 μgCHIKV E2 mRNA are shown in FIGS. 25A-25C. The survival results aretabulated in Table 55.

As shown in Table 54, the 2 μg dose of CHIKV E2 mRNA vaccine gave noprotection post-CHIKV infection challenge when administered via IM or IDin a single dose. However, when provided in two doses, the 2 μg dose ofCHIKV E2 mRNA vaccine provided 80% protection when administered via IMand 100% protection when administered via ID post-CHIKV challenge. Asindicated in Table 55, the 10 μg dose of CHIKV E2 mRNA mouse provided noprotection post-CHIKV challenge when administered via IM or ID in asingle dose. However, administration of CHIKV E2 mRNA via IM or ID usingtwo doses provided 100% protection post-CHIKV challenge.

In all experiments, the negative control mice had a ˜0% survival rate,as did the positive control mice (heat-inactivated CHIKV) which diedprior to CHIKV challenge. Some mice died during the vaccination period.

Example 47: Efficacy of Chikungunya C-E3-E2-6K-E1 Antigen mRNA VaccineCandidate

AG129 mice (n=5 per group) were vaccinated with 2 μg or 10 μg ofMC-3-LNP formulated mRNA encoding CHIKV C-E3-E2-6K-E1 mRNA (SEQ ID NO:388/401). The AG129 mice were vaccinated on either Day 0 or Days 0 and28 via IM or ID delivery. On Day 56 following final vaccination all micewere challenged with a lethal dose of CHIKV. The survival curve, percentweight loss, and health status of the mice vaccinated with 2 μg CHIKVC-E3-E2-6K-E1 mRNA are shown in FIGS. 23A-23C. The survival results aretabulated in Table 56. The survival curve, percent weight loss, andhealth status of the mice vaccinated with 10 μg CHIKVC-E3-E2-6K-E1/E2/E3/C mRNA are shown in FIGS. 26A-26C. The survivalresults are tabulated in Table 57.

As shown in Table 56, the 2 μg dose of C-E3-E2-6K-E1 mRNA vaccineprovided 100% protection post-CHIKV challenge when administered via IMin a single dose and provided 80% protection post-CHIKV challenge whenadministered via ID in a single dose. The 2 μg dose of C-E3-E2-6K-E1mRNA vaccine provided 100% protection post-CHIKV challenge whenadministered via IM or ID in two doses. As shown in Table 57, the 10 μgdose of C-E3-E2-6K-E1 mRNA vaccine provided 100% protection post-CHIKVinfection challenge when administered via IM or ID in either a singledose or in two doses.

In all experiments, the negative control mice had a ˜0% survival rate,as did the positive control mice (heat-inactivated CHIKV) which diedprior to CHIKV challenge. Some mice died during the vaccination period.

Example 48: Summary of Survival Data Using Chikungunya Antigen mRNAVaccine Candidates CHIKV E1, CHIKV E2, and CHIKV C-E3-E2-6K-E1

Table 58 shows the survival data of the mice vaccinated with the CHIKVmRNA antigens used in the studies reported in Examples 45-47.

Example 49: In Vitro Transfection of mRNA-Encoded Chikungunya VirusEnvelope Protein

The in vitro transfection of mRNA encoding Notch and a PBS control wereperformed in 150k HeLa cells/well transfected with 1 μg mRNA+2 μLLF2000/well in a 24 well plate. Lysate containing proteins expressedfrom the CHIKV envelope mRNAs transfected in HeLa cells were collected16 hours post-transfection and then detected by Western blotting with aV5 tag-HRP antibody. The successful detection of a CHIKV envelopeprotein is shown in FIG. 20.

Example 50: Detection of Immunity (Mouse IgG) Against EitherChikungunya-Specific E1, Chikungunya-Specific E2, orChikungunya-Specific E1 and E2 Proteins

Serum samples from mice vaccinated with the CHIKV E1, E2, or E1-E2-E3-Cvaccine described in Examples 45-47 were tested using asemi-quantitative ELISA for the detection of mouse IgG against eitherChikungunya-specific E1, Chikungunya-specific E2, orChikungunya-specific E1 and E2 proteins.

Fifteen groups of five mice were vaccinated via intradermal (ID) orintramuscular (IM) injection with either 2 μg or 10 μg of the candidatevaccine. The vaccines were given to AG129 mice as single or two doses(second dose provided 28 days after the first dose). On day 56, micewere challenged with 1×104 PFU of CHIKV via ID injection in 50 μL volumeand monitored for 10 days for weight loss, morbidity, and mortality.Mice were bled on day 7 and day 28 post-vaccination via the peri-orbitalsinus (retro-orbital bleed). In addition, mice surviving the CHIKVchallenge were bled 10 days post-challenge.

The individual samples were tested for reactivity in a semi-quantitativeELISA for mouse IgG against either Chikungunya-specific E1,Chikungunya-specific E2, or Chikungunya-specific E1 and E2 proteins. Theresults are shown in FIGS. 34-36.

The data depicting the results of the ELISA assay to identify the amountof antibodies produced in AG129 mice in response to vaccination withmRNA encoding secreted CHIKV E1 structural protein, secreted CHIKV E2structural protein, or CHIKV full structural polyprotein C-E3-E2-6k-E1at a dose of 10 μg or 2 μg at 28 days post immunization is shown inFIGS. 34-35. The 10 μg of mRNA encoding CHIKV polyprotein producedsignificant levels of antibody in both studies. The data depicting acomparison of ELISA titers from the data of FIG. 34 to survival in thedata of FIG. 35 left panel is shown in FIG. 36. As shown in the survivalresults, the animals vaccinated with either dose (single or doubleadministration) of mRNA encoding CHIKV polyprotein had 100% survivalrates.

Example 51: Efficacy of Chikungunya Polyprotein (C-E3-E2-6K-E1) mRNAVaccine Candidate

AG129 mice (n=5 per group) were vaccinated with either 10 μg, 2 μg or0.4 μg of MC-3-LNP formulated mRNA encoded CHIKV polyprotein(C-E3-E2-6K-E1) (SEQ ID NO: 388/401). The mice were vaccinated on eitherDay 0 or Days 0 and 28 via IM delivery. In one study, all mice werechallenged on day 56 with a lethal dose of CHIKV following finalvaccination. In another study, all mice were challenged on day 84 with alethal dose of CHIKV following final vaccination. The survival curve,percent weight loss, and health status of the mice vaccinated with 10μg, 2 μg or 0.4 μg mRNA were determined as described previously inExamples 45-47. The survival rates, neutralizing antibodies and bindingantibodies were assessed. Neutralizing antibodies were also identifiedagainst three different strains of CHIKV.

The survival rates of the mice vaccinated with mRNA encoding CHIKVC-E3-E2-6k-E1 is shown in FIG. 37. The data depicts vaccination at adose of 10 μg (left panels), 2 μg (middle panels) or 0.4 μg (rightpanels) at 56 days (top panels) or 112 days (bottom panels) postimmunization. These data demonstrate that a single 2 μg dose of the mRNAvaccine afforded 100% protection for at least 112 days (16 weeks).Following the study out further, the data demonstrated that a single 2μg dose of the mRNA vaccine afforded 100% protection for at least 140days (20 weeks.)

The neutralizing antibody and binding antibody produced in treated miceis shown in FIGS. 38 and 39, respectively. As can be seen in FIGS. 38and 39, the levels of neutralizing antibody were dependent or dose andregimen with the highest titers evident with 10 μg dosed twice (days 0and 28). Plaque reduction neutralization tests (PRNT50 and PRNT80) wereused to quantify the titer of neutralizing antibody for the virus.Antigen-binding Ab was determined by ELISA. The correspondingcorrelation between binding Ab and neutralizing antibodies is shown inthe bottom panels of FIG. 39. Following the study out to 16 weeks showedthat the highest E1 titers were achieved when 10 μg mRNA vaccine wasdosed twice.

The data depicting neutralizing antibodies against three differentstrains of CHIKV is shown in FIG. 40. The neutralizing antibodies weretested against three different strains of CHIKV, African—Senegal (leftpanel), La Reunion (middle panel) and CDC CAR (right panel). FIG. 40shows that the polyprotein-encoding mRNA vaccine elicited broadlyneutralizing antibodies against the three strains tested. Sera werefurther tested against Chik S27 strain (Chikungunya virus (strainS27-African prototype). The data depicting neutralizing antibodiesagainst CHIKV S27 strain is shown in FIG. 41. These data collectivelyshow that the polyprotein encoding mRNA vaccine elicited broadlyneutralizing antibodies against all four strains tested. The vaccineinduced neutralizing antibodies against multiple strains of Chikungunya.The prime and boost with the 10 μg dose produced the most robustneutralizing antibody response followed by the single dose with 10 μg.

Example 52: Transfection of mRNA Encoded CHIKV Structural Proteins

In vitro transfection of mRNA encoding CHIKV structural proteins and PBScontrol were performed in 400 k HeLa cells transfected with 1.25 ug mRNAlipoplexed with 5 ul LF2000/well in 6 well plate. Protein detection inHeLa cell lysate 16 h post transfection was measured. Lysates whichcontain proteins expressed from the CHIKV mRNAs transfected in HeLa werecollected 16 h post transfection. Proteins were detected by WB withanti-Flag or and V5 antibody.

The mRNA encoded CHIKV structural proteins and protein production in theHeLa cell lysate 16 h post transfection was detected.

Example 53: Exemplary CHIKV Polypeptides

The amino acids presented in the Table 48 are exemplary CHIKV antigenicpolypeptides. To the extent that any exemplary antigenic peptidedescribed herein includes a flag tag or V5, or a polynucleotide encodesa flag tag or V5, the skilled artisan understands that such flag tag orV5 is excluded from the antigenic polynucleotide in a vaccineformulation. Thus, any of the polynucleotides encoding proteinsdescribed herein are encompassed within the compositions of theinvention without the flag tag or V5 sequence.

Example 54: Efficacy of CHIKV mRNA Vaccine X Against CHIKV in AG129 MiceStudy Design

Chikungunya virus (CHIKV) 181/25 strain is an attenuated vaccine strainthat was developed by the US Army via multiple plaque-to-plaque passagesof the 15561 Southeast Asian human isolate (Levitt et al.). It is welltolerated in humans and is highly immunogenic. It produces small plaquesand has decreased virulence in infant mice and nonhuman primates. Whenthe attenuated vim s is administered to immunodeficient AG129 mice(lacking the IFN-α/β and γ receptors) the mice succumb to a lethaldisease within 3-4 days with ruffled fur and weight loss (Partidos, etal. 2011 Vaccine).

This instant study was designed to evaluate the efficacy of CHIKVcandidate vaccines as described herein in AG129 mice (Table 59). Thestudy included 14 groups of female 6-8 week old AG129 mice (Table 59).Groups 1-4, 7-8, and 10-15 were vaccinated with CHIKV vaccine X via theintramuscular (IM; 0.05 mL) route on Day 0 and select groups received anadditional boost on Day 28. Control Groups 9 and 16 received vehicle(PBS) only on Days 0 and 28 via IM route (0.05 mL). Regardless ofvaccination schedule, Groups 1-4 and 7-9 were challenged on Day 56 whileGroups 10-16 were challenged on Day 112 using the CHIKV 181/25 strain(stock titer 3.97×10⁷ PFU/mL, challenge dose 1×10⁴ PFU/mouse). For viruschallenge, all mice received a lethal dose (1×10⁴ PFU) of Chikungunya(CHIK) strain 181/25 via intradermal (ID) route (0.050 mL via footpad).All mice were monitored for 10 days post infection for weight loss,morbidity, and mortality. Each mice was assigned a heath score based onTable 51. Mice displaying severe illness as determined by >30% weightloss, a health score of higher than 5, extreme lethargy, and/orparalysis were euthanized with a study endpoint of day 10 post viruschallenge. Test bleeds via retro-orbital (RO) collection were performedon mice from all groups on Days −3, 28, and 56. Mice from Groups 10-16were also bled on Days 84 & 112. Mice that survived challenge were alsoterminally bled on Day 10 post challenge. Serum samples from mice (Days−3, 28, 56, 84, 112 and surviving mice) were kept frozen (−80° C.) andstored until they were tested for reactivity in a semi quantitativeELISA for mouse IgG against either E1, E2 or CHIKV lysate.

Experimental Procedure Intramuscular (IM) Injection of Mice

1. Restrain the animal either manually, chemically, or with a restraintdevice.

2. Insert the needle into the muscle. Pull back slightly on the plungerof the syringe to check proper needle placement. If blood is aspirated,redirect the needle and recheck placement again.

3. Inject appropriate dose and withdraw needle. Do not exceed maximumvolume. If the required volume exceeds the maximum volume allowed,multiple sites may be used with each receiving no more than the maximumvolume.

4. The injection site may be massaged gently to disperse the injectedmaterial.

Intradermal (ID) Injections of Mice

1. Restrain the animal either manually, chemically, or with a restraintdevice.

2. Carefully clip the hair from the intended injection site. Thisprocedure can be done upon animals arriving or the day before anyprocedures or treatments are required.

3. Lumbar area is the most common site for ID injections in all species,but other areas can be used as well.

4. Pinch or stretch the skin between your fingers (or tweezers) toisolate the injection site.

5. With the beveled edge facing up, insert the needle just under thesurface between the layers of skin. Inject the appropriate dose andwithdraw needle. A small bleb will form when an ID injection is givenproperly.

6. If the required volume exceeds the maximum volume allowed, multiplesites may be used with each receiving no more than the maximum volume.

Retro-Orbital Bleeding in Mice

1. Place the mice in the anesthesia chamber and open oxygen line and setto 2.5% purge. Start flow of anesthesia at 5% isoflurane.

2. Once the animal becomes sedate, turn anesthesia to 2.5%-3% isofluraneand continue to expose the animal to the anesthesia. Monitor the animalto avoid breathing becoming slow.

3. Remove the small rodent from anesthesia chamber and place on its backwhile restraining with left hand and scruff the back of the animal'sneck, so it is easy to restrain and manipulate while performing theprocedure with the right hand.

4. With a small motion movement, place the capillary tube in the cornerof the animal's eye close to the nostril, and rotate or spin theHematocrit glass pipette until blood start flowing out. Collect theappropriate amount of blood needed into the appropriate labeled vial.

5. Monitor the animal after retro-orbital bleeding is done for at least10-15 seconds to ensure hemostasis.

6. Place the animal back to its original cage and monitor for any otherproblems or issues caused while manipulating animal due to theprocedure.

Observation of Mice

1. Mice were observed through 10 days post infection (11 days total,0-10 days post infection).

2. Mice were weighed daily on an Ohause scale and the weights arerecorded.

3. Survival and health of each mouse were evaluated once time a dayusing a scoring system of 1-7 described in Table 51.

Infection

On either Day 56 (Groups 1-4, 7-9) or Day 112 (Groups 10-16) groups of 5female 6-8 week old AG129 mice were infected via intradermal injectionwith 1×10⁴ PFU/mouse of the 181/25 strain of Chikungunya diluted in PBS.The total inoculation volume was 0.05 mL administered in the rearfootpad of each animal. Mice were anesthetized lightly using 2-5% v/v ofisoflurane at ˜2.5 L/min of 02 (VetEquip IMPAC6) immediately prior toinfection.

Dose Administration

In this study mice were administered 0.04 μg, 2 μg, or 10 μg of variousformulations of the CHIKV vaccine X or vehicle alone (PBS) on either Day0 or on Days 0 and 28 via the intramuscular route (0.05 mL). Thematerial was pre-formulated and diluted in PBS by IBT prior to dosing.

Results

Mice were immunized once (Day 0) or twice (Days 0 & 28) with either 0.04μg, 2 μg, or 10 μg of Chikungunya mRNA vaccine X and were challengedwith CHIKV strain 181/25 on either Day 56 (Groups 1-4, 7-9) or on Day112 (Groups 10-16). Mice were monitored for a total of 10 days postinfection for health and weight changes. Mice that received either 2 μgor 10 μg of the CHIKV mRNA vaccine X either once (Day 0) or twice (Days0 and 28) were fully protected (100%) regardless of whether the micewere challenged 56 days or 112 days after the initial vaccination (FIGS.27A-27B, Table 44). Mice receiving 0.04 μg of the CHIKV mRNA vaccinewere not protected at all from lethal CHIKV infection. This efficacydata is supported by the health scores observed in the vaccinated micein that the protected mice displayed little to no adverse health effectsof a CHIKV infection (FIGS. 29A-29B). Weight loss is not a strongindicator of disease progression in the CHIKV AG129 mouse model (FIGS.28A-28B).

Mice immunized with the CHIKV mRNA vaccine X showed increased antibodytiters against CHIKV E1, E2 and CHIKV lysate as compared to the vehicleonly (PBS) treated groups. Serum binding against the virus lysateyielded the highest antibody titers for all vaccinated groups (FIGS.30A-30C, 31A-31C, 32A-32C, 33A-33C). Overall, the antibody titers weredose dependent with the highest titers observed in serum from micevaccinated with 10 μg of CHIKV mRNA vaccine X while the lowest titerswere observed in serum from mice vaccinated with 0.04 μg of the CHIKVmRNA vaccine X. Similarly, higher titers were observed in serum frommice vaccinated twice (Days 0 and 28) as compared to serum from micevaccinated only once (Day 0). Serum obtained on Day 112 post initialvaccination still yielded increased antibody titers in mice thatreceived either 10 μg or 2 μg of CHIKV mRNA vaccine X (FIGS. 32A-32C).

Serum from mice groups 10-16, 112 days post immunization were alsotested in a Plaque Reduction Neutralization Test (PRNT). Serum from eachmice was diluted from 1/20 to 1/40960 and assessed for its ability toreduce CHIKV plaque formation. The results were shown in Table 64.

Example 55: Immunogenicity of Chikungunya Polyprotein (C-E3-E2-6K-E1)mRNA Vaccine Candidate in Rats

Sprague Dawley rats (n=5) were vaccinated with 20 μg of MC-3-LNPformulated mRNA 30 encoded CHIKV polyprotein (C-E3-E2-6K-E1) (SEQ ID NO:388/401). The rats were vaccinated on either Day 0 or Days 0 and 14 orDays 0, 14 and 28 via IM delivery. Sera were collected on days −3, 14,28 and 42 for ELISA testing. FIG. 42 demonstrated that there was atleast a two log increase in antibody titer against CHIKV lysate post 3rdvaccination with the mRNA vaccine in normal rats.

Example 56: Evaluation of T Cell Activation of Chikungunya P 5Polyprotein (C-E3-E2-6K-E1) mRNA Vaccine Candidate

C57BL/6 mice (n=6 experimental group; n=3 control group) were vaccinatedwith 10 μg of MC-3-LNP formulated mRNA encoded CHIKV polyprotein(C-E3-E2-6K-E1) (SEQ ID NO: 388/401). The mice were vaccinated on eitherDay 0 or Days 0 and 28 (boost) via IM delivery. Sera was collected ondays 3, 28 and 42 for ELISA testing. Animals were sacrificed on day 42and spleens were harvested for immunological evaluation of T cells.Splenic cells were isolated and analyzed by FACS. Briefly, spleens wereremoved, cells isolated, and stimulated in vitro with immunogenicpeptides found within either C, E1, or E2 region of CHIKV that are knownto be CD8 epitopes in B6 mice. The readout for this assay was cytokinesecretion (IFN-gamma and TNF-alpha), which reveals whether the vaccineinduced antigen-specific T cell responses. No CD8 T cell responses weredetected using the E2 or C peptide (baseline levels of IFN-gamma andTNF-alpha), whereas there was a response to the E1-corresponding peptide(average of about 0.4% IFN-gamma and 0.1% TNF). The peptides were usedto stimulate T cells used in the study were E1=HSMTNAVTI (SEQ ID NO:414), E2=IILYYYELY (SEQ ID NO: 415), and C=ACLVGDKVM (SEQ ID NO: 416).

FIG. 43 shows that the polyprotein-encoding CHIKV polyprotein vaccineelicited high antibody titers against the CHIKV glycoproteins. FIGS. 44and 45A-45B show T cell activation by E1 peptide.

Example 57: Proof-of-Concept of Immunogenicity in Non-Human Primates

The mRNA vaccine was tested in Cynomolgus monkey subjects (n=3 perexperimental group, n=3 negative control). Subjects were given anintramuscular (IM) immunization of 25 μg or 75 μg of the vaccine on day0 (prime), day 28 (boost), and day 56 (boost). The negative controlgroup was administered 75 μg of non-translated irrelevant mRNA (NTIX).The readout for this experiment was serum antibody titers (binding andneutralizing) and a CHIKV-specific T cell response.

As shown in FIG. 46, the vaccine induced a robust antibody response. Aresponse was detected after the priming dose, and then increased withthe boost, and increased slightly more following the third immunization.Both the 25 μg and 75 μg vaccine groups were immunogenic, and there wasa small dose response. Neutralizing titers were a few fold lower thanthose seen in mice, but were still robust.

FIG. 47 shows a robust CD4 response in response to the vaccine. Day 35 Tcells, measured one week after the second immunization, were assayed.The peptide pool consisted of 15 mers overlapping by 11. The responsewas measured through peptide stimulation, followed by intracellularcytokine staining and flow cytometry. A CHIKV-specific CD4 T cellresponse was detected, mainly in IL-2 and TNFα. There was a minimal CD8response as well.

Each of the sequences described herein encompasses a chemically modifiedsequence or an unmodified sequence (no modified nucleotides), whichincludes no nucleotide modifications.

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EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the disclosure described herein. Such equivalents areintended to be encompassed by the following claims.

All references, including patent documents, disclosed herein areincorporated by reference in their entirety.

LENGTHY TABLES The patent application contains a lengthy table section.A copy of the table is available in electronic form from the USPTO website(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20190008946A1).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

1-147. (canceled)
 148. A method of inducing an immune response in a subject, the method comprising administering to the subject an immunogenic composition comprising a ribonucleic acid (RNA) polynucleotide having an open reading frame encoding a Zika virus (ZIKV) prME protein that comprises an amino acid sequence that has at least 95% identity to the amino acid sequence of SEQ ID NO: 203, wherein the immunogenic composition is administered in an effective amount to produce an antigen-specific immune response in the subject.
 149. The method of claim 148, wherein the antigen specific immune response comprises a T cell response or a B cell response.
 150. The method of claim 148, wherein the subject is administered a single dose of the immunogenic composition.
 151. The method of claim 148, wherein the subject is administered a booster dose of the immunogenic composition.
 152. The method of claim 148, wherein the immunogenic composition is administered to the subject by intradermal injection or intramuscular injection.
 153. The method of claim 148, wherein an anti-ZIKV prME antibody titer produced in the subject is increased by at least 1 log and/or at least 2 times relative to a control. 154.-156. (canceled)
 157. The method of claim 153, wherein the control is selected from an anti-ZIKV prME antibody titer produced in a subject who has not been administered a vaccine against the virus, an anti-ZIKV prME antibody titer produced in a subject who has been administered a live attenuated vaccine or an inactivated vaccine against the virus, an anti-ZIKV prME antibody titer produced in a subject who has been administered a recombinant protein vaccine or purified protein vaccine against the virus, and an anti-ZIKV prME antibody titer produced in a subject who has been administered a VLP vaccine against the virus. 158.-185. (canceled)
 186. The method of claim 148, wherein the RNA polynucleotide is formulated in a lipid nanoparticle that comprises a molar ratio of 20-60% ionizable cationic lipid, 5-25% non-cationic lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid.
 187. The method of claim 148, wherein the ZIKV prME protein comprises the amino acid sequence of SEQ ID NO:
 203. 188. The method of claim 148, wherein the RNA polynucleotide further encodes a 5′ terminal cap, 7 mG(5′)ppp(5′)NlmpNp.
 189. The method of claim 148, wherein at least 80% of the uracil in the open reading frame has a chemical modification selected from N1-methyl-pseudouridine and N1-ethyl-pseudouridine.
 190. The method of claim 189, wherein the chemical modification is in the carbon 5-position of the uracil.
 191. The method of claim 148, wherein the efficacy of the immunogenic composition is at least 70%, relative to unvaccinated subjects, following a single dose of the immunogenic composition.
 192. The method of claim 148, wherein the effective amount is sufficient to produce detectable levels of ZIKV prME protein as measured in serum of the subject at 1-72 hours post administration.
 193. The method of claim 148, wherein the effective amount is sufficient to produce a 1,000-10,000 neutralization titer produced by neutralizing antibody against the ZIKV prME protein as measured in serum of the subject at 1-72 hours post administration.
 194. The method of claim 148, wherein the effective amount is a total dose of 25 μg-400 μg.
 195. The method of claim 186, wherein the ionizable cationic lipid comprises the following compound:


196. The method of claim 148, wherein the RNA polynucleotide has an open reading frame that comprises a nucleotide sequence that has at least 90% identity to the nucleotide sequence of SEQ ID NO:
 139. 197. The method of claim 196, wherein the RNA polynucleotide has an open reading frame that comprises the nucleotide sequence of SEQ ID NO:
 139. 